Improved biosynthetic wound and burn dressing with silver-based broad antimicrobial activity

RELATED APPLICATIONS

This application is a Continuation-in-Part of PCT Application PCT/US20/37962 filed June 16, 2020, and that application is included here by reference in its entirety.

FIELD OF THE INVENTION

This invention relates to primary dressings and bandages for both acute and chronic wounds, particularly for burns.

BACKGROUND OF THE INVENTION

Background for a primary wound dressing of skin substitute containing broad antimicrobial activity: Over the past 20 years numerous wound dressings containing silver have been introduced to the wound care market. Examples are: Acticoat, Tegaderm w/ Ag, Aquacel-Ag, Maxorb Extra Ag, Melgisorb, and so on. Silver in wound dressings is used in various forms, usually as a silver salt and reported as ppm or grams added per unit area/volume of the dressing.

PermeaDerm has many desirable properties which are described as essential for an Ideal Skin Substitute or primary wound dressing: adherent, long shelf-life at room-temp, transparent, moist healing, conformable, flexible, non-toxic, strong, low-cost, non-biodegradeable membrane oxygenator, sterile, low dressing change and low pain. Adding silver to PermeaDerm provides an important property - broad antimicrobial activity. Silver, a heavy metal can damage good and bad cells. The mode of action is generally protein denaturation.

The objective here is to include a silver salt in the 3D structure of PermeaDerm at a minimum concentration that effectively kills microbes over a period of time. This strategy has intent to kill either endogenous/exogenous microbes inside the 3D structure of PermeaDerm while minimizing damage to autogenous cells and tissue that grow onto the 3D structure of PermeaDerm. The US Army Medical Material Activity (USAMMDA) and US Army Institute of Surgical Research (USAISR) has expressed interest in PermeaDerm for use on the battlefield to reduce pain associated with blast injury to soldier’s extremities so they can continue with their battle duties in the field with minimum discomfort. PermeaDerm possesses properties important to the USAMMDA and USAISR which are listed above where adherence and pain reduction are critical. The addition of broad antimicrobial activity would satisfy the US Army’s need for a primary wound dressing or skin substitute with optimal properties for their soldiers. In the domestic wound care market; closure with PermeaDerm has been proven successful for both acute and chronic wound management.

The incidence of infection complication is low due to the variable porosity of PermeaDerm and its ability to minimize wound fluid accumulation beneath PermeaDerm. Addition of a silver salt (silver citrate) adds broad antimicrobial activity to the wide range of properties and benefits already provided by PermeaDerm.

Bacterial contamination is possible from two sources: endogenous to the wound or exogenous to the wound. Major risk factor for patients with diabetic foot ulcers is infection, gangrene and subsequent amputation. Cost of care for lower leg amputation is substantial at $52,000 per year for reimbursement. Most clinical infections are from microbes endogenous to the wound or microbes already present in the wound. PD’s silicone membrane is 100% impermeable to exogenous microbes except at a slit opening. PD is 99% occlusive therefore a great barrier to exogenous microbes outside the wound.

Relevant Prior Art - the following list is a summary of prior art for the science of using silver for wound care, with some comments:

US201 60030476 VACHON DAVID J et al. “Compositions, methods and devices for promoting wound healing and reducing infection.” Similar objective to kill microbes contaminating the wound while minimizing damage to autogenous tissue. Generally describes numerous materials that silver species can be incorporated into. Does speak to silver concentration effect in killing microbes and autogenous skin cells. Does not describe a gelatin/aloe material for combining silver with. And does not speak to shelf-life properties of a wound dressing and how a silver-gelatin salt (like Kodak film) would improve room-temperature shelf-life. US20070009580 DICOSMO FRANK et al. “Non-adhesive hydrogels” Specific to hydrogels which are different than our bilaminate of silicon/nylon with bio-coat of gelatin/aloe onto the 3D structure of the present invention. US10350219 KARANDIKAR BHALCHANDRA M. “Antimicrobial compositions and methods of making the same”. Specific to hydrogels different than our bilaminate of silicon/nylon with bio-coat of gelatin/aloe onto the 3D structure of PermeaDerm.

US6808738 DITIZIO VALERIO et al. “Method of making anti-microbial polymeric surfaces “ Specific to hydrogels which is different than our bilaminate of silicon/nylon with bio-coat of gelatin/aloe onto the 3D structure of The present invention.

US9937156 SMALL ERIC M et al. “Modulation of mrtf-a activity in pathologic fibrosis and wound healing”. Specific to acrylate-modified silicon which is different than our bilaminate of silicon/nylon with bio-coat of gelatin/aloe onto the 3D structure of the present invention.

US201 80008742 HOGGARTH ANDREW et al. “ Composition for a wound dressing”. Fairly general wound dressing which is different than our bilaminate of silicon/nylon with bio-coat of gelatin/aloe onto the 3D structure of the present invention.

US201 2031 8262 LEE ETHAN et al. “Pyrvinium wound treatment methods and devices”. Fairly general wound dressing which is different than our bilaminate of silicon/nylon with bio-coat of gelatin/aloe onto the 3D structure of the present invention.

SUMMARY OF THE INVENTION

The present invention is a new and improved approach to providing a bandage that addresses burn injuries. The state-of-art bandages do not possess the new features of the present invention, directed to allowing a measured amount of porosity, including essentially zero porosity, and a lower cost to produce combined with shorter healing/rehabilitation times .

The goal was to create a more effective biosynthetic bandage for burns management. . A hydrophilic/hygroscopic moist, but not wet, wound healing environment which enhances healing.

Wound sites have variable amounts of exudate/transudate/plasma present, from dry to weepy. The clinician must cleanly debride the wound, close it and manage wound healing in a moist but not wet environment to achieve optimal results ie for both acute and chronic wounds. This is particularly difficult with complicated structures like hands and feet.

The present invention provides a dressing that possesses all the properties and attributes of an ideal skin substitute and, in addition, has ‘variable porosity’ controlled by the clinician from zero porosity to what the wound requires. The present invention enables the clinician to move the fluid exuding from the wound through the primary dressing into an absorbent secondary dressing without disturbing the kinetics of healing or causing pain to the patient.

The present invention will have a single layer of biologicals comprised of a hypoallergenic BSE free USP Pharmaceutical grade porcine gelatin, pure Aloe or Aloesin, pure Aloe and BSE free porcine gelatin, or a mixture of pure Aloe, BSE free gelatin and ECM interact with the wound to stimulate the rate of healing while adherent to the wound. The bio-coat layer is deposited directly on the nylon side of the “variable porosity” silicone/nylon surface and is stable for over five years at room temperature.

In vitro, the Aloe component has been demonstrated to cause a variety of cells to attach and proliferate; as well as increase synthesis of collagen and alpha smooth muscle actin. ECM (extra-cellular matrix) may be added to the biologicals described above and is a mixture from human fibroblasts that is known to cause rapid cell proliferation and tissue growth. Previous wound dressings and skin substitutes, as taught in US Pat. 7,815,931 contain gelatin, a pure Aloe component, chondroitin 4 & 6 sulfate, and vitamin C & E. In contrast the current dressing will have two layers of biologicals applied in separate spraying operations as described above. The first coat will contact the wound after the second coat of hypoallergenic bovine spongiform encephalopathy (BSE) - free United States Pharmaceutical (USP) - grade porcine gelatin interacts with fibrin in the wound to achieve early adherence, The second coat of biologicals stimulates the healing process during the interval where the dressing invention is in contact with the wound and is stable requiring 100 degree water for 30 minutes to remove from the “variable porosity” silicone/nylon surface.

Human wounds vary in the following ways depending on location: relatively flat, convex, or concave surface; and vary in movement from minimal to a lot (diaphragm). Most difficult location to achieve adherence are convex wounds where there is movement (axilla, upper abdomen, etc.). Irregular depth wounds such as ulcers (DFU, VLU - diabetic foot ulcer and venous leg ulcer) require prompt closure requiring excellent compliance, stretchability or elongation.

If slow-healing ulcers (DFU, VLU, PU - pressure ulcer) are not closed promptly infection complications often occur and potential loss of limb and compromise of lifestyle is at risk. Clinical preference is current PDC (average thickness is 0.0080” with STDEV of 0.0008”) for protection of widely meshed autograft where maximization of healing (definitive closure) of the interstices is desired. Too much adherence (greater than 50 gm per sq. cm. at 5 hours or 0.0048” thick) will slow interstices healing of a widely meshed autograft.

A thicker 3D structure (average thickness 0.0080” with STDEV 0.0008”) for PDG (PermeaDerm® glove) provides additional advantages over normal thickness PDG with respect to greater circumferential elongation and fit in the web spaces giving the clinician greater room for error during the fitting process. Once properly fitted, PDG will enable rapid rehabilitation without pain to regain full range of motion and will minimize interference with the healing process yielding excellent aesthetic and functional results. PDB and PDG (PermeaDerm® Chronic) would clinically benefit from both greater adherence and greater elongation/conformability.

There is a Regulatory advantage (FDA approval) based on use of same material (15/1 denier virgin nylon filament) to increase thickness of 3D structure of PermeaDerm® product as opposed to use of a different denier of virgin nylon filament (example 15/2, 15/3, 18/3 denier). Estimated thickness using SEM: 15/1 denier about 0.0069” and 18/3 denier about 0.014”. 3D structure of 18/3 denier is about 103% (more than double) thicker than 15/1 denier.

PermeaDerm has been proven to possess a room temperature shelflife of at least five years. PermeaDerm with silver is expected to have a shelf-life of more than five years at room temperature based on known properties of Kodak film which contain gelatin-silver as the active component of photographic film. “At a room temperature of 24°C (75°F) or colder, with the relative humidity no higher than 60 percent, and if the print was properly processed, the dyes should remain relatively stable for about 20 years.”

There is a further goal to Incorporate silver ion into the 3D matrix of the present invention at the lowest concentration where antimicrobial activity is effective (kills 100,000 microbes). We want a 3 uL (2 ^nd biocoat of Silver Citrate/Citric Acid solution Spray Solution) to dispense 0.54 ug of silver ion or 0.885 ug of silver citrate monohydrate per each square cm surface of PermeaDerm 3D which faces the wound.

Key tactic here is minimizing the interaction of silver with Aloe and gelatin, therefore we use a two-step formulation process:

First step is formulating a stable biocoat of Aloe/gelatin and subsequent drying the same - it is stable because of PermeaDerm’s real-time shelf-life properties at room-temperature for at least five years. Essentially the only biodegradable component of PermeaDerm over time is Aloe and gelatin, the biological components of the product (less than 2% of the PermeaDerm

- over 98% of PermeaDerm is the stable synthetic components - silicone/nylon membrane).

Second step is application of a higher concentration of silver citrate to the 3D structure over the top of the standard Aloe/gelatin biocoat - defined in the Silver PermeaDerm Formulation.

Wounds closed with a biocoated silicone/nylon membrane needs to be adherent, without adherence it will not work.

Adherence in wounds can be compromised by fluid accumulation beneath the PermeaDerm - solution is various slits that can be enlarged by clinician to enable wound fluid to move from beneath the PermeaDerm into an absorbent secondary dressing which can be periodically replaced without compromise of adherence or wound healing

Use of PermeaDerm is never compromised by accumulation of gases (oxygen, carbon dioxide, water) beneath PermeaDerm. The area of PermeaDerm containing no slits is close to 99%. Gases can dissolve into solid silicone and diffuse through based on a concentration gradient:

Diffusion: Diffusion is the gradual mixing of gases due to the motion of their component particles even in the absence of mechanical agitation such as stirring driven by gases at a higher concentration always moving to a lower concentration. A membrane oxygenator separates blood from air. Typically the oxygen concentration in air is higher than in blood and the oxygen always moves toward a lower concentration - from air to blood.

Silicone : chemically known as polydimethylsiloxane (PDMS), is among the most gas permeable dense polymeric membrane materials available . Gases permeate silicone by a solution / diffusion mechanism, whereby the rate of gas permeation is directly proportional to the product of solubility of the gas, and the rate of diffusion of the dissolved gas in silicone. The permeability coefficient is a parameter defined as the transport flux of a gas (rate of gas permeation per unit area), per unit transmembrane driving force, per unit membrane thickness.

The following are two wound oxygenation approaches in the relevant literature. They are easily distinguishable from the present invention: OxyBand Wound Dressing™ is the only FDA-approved wound dressing proven to continuously deliver pure oxygen to damaged tissue for up to 5 days. OxyBand Wound Dressing™ is a patented medical technology with a directionally permeable gas-emitting reservoir inside of the dressing. OxyBand Wound Dressing™, Food and Drug Administration FDA 510(k), K043063, provides oxygen to wounds for up to 5 days. The oxygen is pre packaged and in a proprietary method, diffused into the patented reservoir system inside of the OxyBand Wound Dressing™. The pre-filled multi-layer, OxyBand Wound Dressing™, comes pre-filled with high levels of oxygen between the layers. OxyBand Wound Dressing™ incorporates a barrier layer that holds the oxygen, in the vicinity, of the wound, and a permeable or porous layer that allows oxygen to diffuse into the wound. The dressing acts like an oxygen reservoir, allowing the wound to utilize as much oxygen as needed, and continues to supply oxygen on demand to the wound as the wound consumes oxygen from the wound fluid.

Oxygen generating wound dressing US Patent 5,792,090 Aug 11, 1998. Abstract: The healing of surface wounds, including burns, is facilitated by increasing the wound oxygen tension through the application of an oxygen-generating wound dressing which renewably and non-sustainingly chemically generates oxygen. The wound dressing contains an oxygen permeable membrane and an oxygen supply solution reservoir adapted to receive an aqueous liquid capable of supplying oxygen through chemical reaction. Preferably, the aqueous liquid contains hydrogen peroxide and the reservoir contains an immobilized solid hydrogen peroxide decomposition catalyst such as manganese dioxide. The solid catalyst may be contained within a hydrogel, an open-celled polymeric foam, or a biosynthetic foam such as a collagen foam. The oxygen-generating wound dressing may be applied over an intermediate hydrogel occlusive wound covering such as an alginate.

DETAILED DESCRIPTION

The present invention is similar in composition to earlier skin substitutes in that they each have a thin silicone component and an underlying 3D thin knitted nylon component. The present invention differs from its ancestors in that it has “variable porosity” controlled by the clinician; the slit width in the thin silicone will be essentially zero (with no stretch, in relaxed mode) to a higher porosity (proportional to the stretch applied).

In addition, the present invention differs from prior art in the composition of biological coatings applied to both components and how these coatings interact with the wound over time.

The preferred embodiment of the invention designed for burns, a regular pattern with slits on the silicone surface parallel to each other in the same row is presented. The slits made in the silicone are approximately. 0.125” long with a space of 0.50”, between the slits; off-set parallel rows of slits are 0.25” apart.

In this configuration the silicone/nylon membrane can be stretched in a direction perpendicular to the slit orientation and the slits will open. Porosity therefore increases proportionally to the amount of stretch applied. Obviously, there is a maximum amount of stretching of the preferred embodiment before the dressing fails. IFU (instructions for use) describe a maximum of 20% elongation, well below the breaking point.

The use of the present invention has a large benefit because it is stable on the wound and possesses biologicals that aid in the healing process.

The present invention will have a first layer of biologicals comprised of a hypoallergenic BSE free USP Pharmaceutical grade porcine gelatin, pure Aloe or Aloesin, pure Aloe and BSE free porcine gelatin, or a mixture of pure Aloe, BSE free porcine gelatin and ECM interact with the wound to stimulate the rate of healing while adherent to the wound.

The biologicals are applied to the knitted nylon side of the present embodiment. Optionally, the biologicals can be applied to the silicone layer as well.

The preferred embodiment is the Gen3 PermeaDerm with average thickness of 0.0080” and STDEV of 0.0008” which results in Optimal adherence.

Improved PermeaDerm product with addition of silver citrate:

Stability at room-temp: Current Biocoat of PermeaDerm has been proven to possess a room temperature shelf-life of at least five years. PermeaDerm with silver is expected to have a shelf-life of more than five years at room temperature based on known properties of Kodak film which contain gelatin-silver as the active component of photographic film. “At a room temperature of 24°C (75°F) or colder, with the relative humidity no higher than 60 percent, and if the print was properly processed, the dyes should remain relatively stable for about 20 years. (This dye stability estimate is based on EASTMAN Color Print Film, manufactured in 1990.”)

Clinical and US Army need for a skin substitute with broad antimicrobial activity: Over the past 20 years numerous wound dressings containing silver have been introduced to the wound care market. Examples are: Acti- coat, Tegaderm w/ Ag, Aquacel-Ag, Maxorb Extra Ag, Melgisorb, and so on. PermeaDerm has many desirable properties which are described as essential for an Ideal Skin Substitute: adherent, long shelf-life at room-temp, transparent, moist healing, conformable, flexible, non-toxic, strong, low- cost, membrane oxygenator, sterile, low dressing change, and low pain. Adding silver to PermeaDerm provides an important property - broad antimicrobial activity.

The present invention also claims superior oxygenation for wounds, due to its structure. Gases permeate silicone by a solution / diffusion mechanism, whereby the rate of gas permeation is directly proportional to the product of solubility of the gas, and the rate of diffusion of the dissolved gas in silicone. The permeability coefficient is a parameter defined as the transport flux of a gas (rate of gas permeation per unit area), per unit transmembrane driving force, per unit membrane thickness. The permeability coefficient for various gases and vapors in silicone is presented in the table below (Membrane Basics I PermSelect-MedArray). Permeability coefficients: oxy- gen 600, Carbon dioxide 3,250 and water 36,000. The gases of clinical interest are oxygen, carbon dioxide and water.

Diffusion rate: The rate of diffusion (diffusion of gases through silicone membranes - Bing) through the silicone membrane is inversely proportional to the thickness of the membrane, any factor that increases the thickness to more than two to three times normal can interfere significantly with normal respiratory exchange of gases.

Bilaminate membranes of silicone/nylon: The art of making strong and thin membranes of silicone and nylon were accomplished in 1978 with invention of Biobrane and continued with AWBAT 2008, and PermeaDerm 2013. The method is a precision knife blade of a dilute dispersion of dimethyl-silicone in xylene onto a perfectly flat surface, then combined with a precise knitted nylon structure, and then cured to make the silicone a solid. Gases (oxygen, carbon dioxide and water) rapidly dissolve in the solid silicone membrane as they move from a higher concentration to a lower concentration. The achievement of silicone/nylon membranes with thicknesses of less than one mil enhances the ability of gases to diffuse through the thin, strong solid silicone membrane. The clinical significance of higher oxygen and lower carbon dioxide is apparent faster healing of chronic wounds which have defective vascular supply.

Use of SEM to illustrate the thickness of the PermeaDerm solid silicone membrane - about 0.0003” thick or extremely thin which functions well as a membrane oxygenator (attached PD Thickness). The US Army Medical Materiel Development Activity (USAMMDA) and U.S. Army Institute of Surgical Research (USAISR) have expressed interest in PermeaDerm for use on the battlefield to reduce pain associated with blast injury so they can continue with their battle duties with minimum discomfort. PermeaDerm w/ silver would be ideal to quickly close battlefield wounds until the soldier can be treated later in a hospital. Properties important to the USAMMDA and USAISR are listed above plus antimicrobial activity. In the domestic wound care market; closure with PermeaDerm is already successful for acute and chronic wound management. PermeaDerm w/ silver adds another very important benefit: broad antimicrobial activity.

PermeaDerm has been proven to possess a room temperature shelf-life of at least five years. PermeaDerm with silver is expected to have a shelf-life of more than five years at room temperature based on known properties of Kodak film which contain gelatin-silver as the active component of photographic film. “At a room temperature of 24°O (75°F) or colder, with the relative humidity no higher than 60 percent, and if the print was properly processed, the dyes should remain relatively stable for about 20 years.”

The formulation for making Silver PermeaDerm is a two-step biocoat process using the BioDot XYZ spray system. The first step is our Standard method of adding a gelatin/Aloe biocoat (6 microliter per sq. cm. of PermeaDerm) to the 3D surface of PermeaDerm and dried per normal protocol. After the application of the 1 ^st gelatin/Aloe biocoat and drying (normal protocol) the 2 ^nd biocoat of a silver citrate/citric acid solution (3 microliter per sq. cm. of PermeaDerm) is applied to the 3D surface of PermeaDerm and dried per normal protocol.

Methods, Results and Conclusions: Desire 3 uL (2 ^nd biocoat of Silver Citrate/Citric Acid

Desire 3 uL (2 ^nd biocoat of Silver Citrate/Citric Acid solution Spray Solution) to dispense 0.54 ug of silver ion or 0.885 ug of silver citrate monohydrate per each square cm surface of PermeaDerm 3D which faces the wound. This concentration is enough to kill 100,000 colony forming units per ml Staphylococcus epidermis at 7, 14 and 21 days. The solution appears stable, has minimal turbidity (as measured at 660 nm) and the silver ion concentration is constant over at least a one month. The formulation for making Silver PermeaDerm is a two-step biocoat process using the BioDot XYZ spray system. The first step is our Standard method of adding a gelatin/Aloe biocoat (6 microlite per sq. cm. of PermeaDerm) to the 3D surface of PermeaDerm and dried per normal protocol. After the application of the 1 ^st gelatin/Aloe biocoat and drying (normal protocol) the 2 ^nd biocoat of a silver citrate/citric acid solution (3 microliter per sq. cm. of PermeaDerm) is applied to the 3D surface of PermeaDerm and dried per normal protocol.

Procedure for Preparation and Stability Characterization of 250 ml_ of Silver Citrate/Citric Acid Solution Aim: to make 250 ml_ of Silver Citrate Solution (to deliver 5x the amount (as opposed to formulation on October 27, 2020) of silver ion (0.54 ug per sq. cm in 3 uL)

Record weight (+/- 1 %) of:

1 . Beaker plus magnetic stirring bar

2. Sterile water 250 g

3. Silver citrate 74.15 mg

4. USP Citric Acid 56.0 gm

Procedure (follow standard Trelleborg Batch Record Shop Oder instructions and record keeping)

1. Add 200 ml_ of H ₂ O to beaker w/ stirring bar. Keep constant stirring.

2. Gradually add 56.00 g of citric acid until fully dissolved

3. Gradually add 74.15 mg of silver citrate until fully dissolved

4. When dissolved, add to 250 ml_ volumetric flask and bring to 250 ml_ volume with distilled water.

When complete, store at refrigerated temperature (4°C ± 1 °C) for 30 days. After 30 days, filter and weigh filtrate to determine any precipitation. Also, observe solution for cloudiness or any other signs of precipitation of the silver citrate. Preferred Embodiment:

Methods, Results and Conclusions: Desire 6 uL of Silver PermeaDerm BioCoat Spray Solution to dispense 0.108 ug of silver ion or 0.177 ug of silver citrate monohydrate per each square cm surface of PermeaDerm 3D which faces the wound. This concentration is enough to kill 100,000 colony forming units per ml Staphylococcus epidermis at 7, 14 and 21 days. The solution appears stable, has minimal turbidity (as measured at 660 nm) and the silver ion concentration is constant over at least a one month.

Gen 1 : 1 .0 sq. cm. of Genl (average thickness 0.0043” or 0.0109 cm) has a volume of 0.0109 ml containing 0.108 ug of silver ion or a concentration of 9.908 ppm silver. This provides 9.908 ug silver ion per ml of 3D structure.

Gen 3: 1 .0 sq. cm. of Gen3 (average thickness 0.0080” or 0.0203 cm) has a volume of 0.0203 ml containing 0.108 ug of silver ion or a concentration of 5.32 ppm silver. This provides 5.32 ug silver ion per ml of 3D structure.

Procedure: to make 100 ml of Silver PermeaDerm Bio-coat Spray Solution (to make 250 mi as opposed to 100 ml use 2.5 x amounts defined below)

Record weight (+/- 3%) of:

1. Beaker plus magnetic stirring bar

2. Sterile water 100 gm

3. Silver citrate/citric acid solution 0.45 gram 4. MedullaPro (gelatin) 0.5 gm

5. lmmuno-10 (Allo) 1 .0 gm

Procedure - for making silver citrate/citric acid solution:

1 . Dissolve 2.24 grams of USP Citric Acid in 9.0 ml_s of distilled water.

2. Add 72.0 mg of Silver Citrate Hydrate and mix to dissolve.

3. Add above mixture to 10 ml_ volumetric flask and add distilled water to volume.

Procedure (follow standard Trelleborg Batch Record Shop Oder instructions and record keeping)

1. Add 100 ml of H2O to beaker w/ stirring bar and bring temperature to 60 degrees C

2. Gradually add 0.5 mg of MedullaPro (gelation) until fully dissolved

3. Gradually add 1 .0 mg of lmmuno-10 (Allo) until fully dissolved

4. Discontinue heating the solution

5. When temperature of solution has cooled about 10 degrees C: gradually add 0.45 grams of silver citrate/citric acid solution until fully dissolved

6. Correct for loss of H2O - same procedure as described in current Trelleborg “Batch Record Shop Order” Tests of the Efficacy of Preferred Embodiment

TEST 1

Procedure:

The Silver Citrate PRDM C 5” x 57Lot SO 63331 RD pads were digested in standard manner and analyzed by ICPMS to determine silver content per cm2 in the 5” x 5” pads. Result:

Test 1 : 0.000580 ug Silver I cm ² Test 2: 0.000580 ug Silver I cm ² Test 3: 0.000414 ug Silver I cm ² Test 4: 0.000580 ug Silver/ cm ² The expected amount of silver I cm ² on the pads tested is 0.108 ug Silver/ cm ² . The recovery was approximately 0.5%% of the expected amount using this method. We were not sure if the low recovery was due to the sample being low in silver or if the testing methodology was in error. Other digestion methods produced similar low silver concentration results in the pads tested (Tests 5 to 9 results similar or less than tests 1-4)). After the investigation further into the formulation components of the pads, we assumed that the silver was likely precipitated as silver chloride in the pads and had to be digested and treated as such in the analytical procedure. The method settled on was an ashing method to incinerate all of the organics and to ostensibly remove chloride by conversion into gaseous chlorine and then acid digestion. This procedure was much better and produced the following amount of silver:

Results:

Test 10: 0.0955 ug Silver/ cm ² . This is 88% of the expected silver in the sample tested.

Test 11 : Spiked 90ug of Silver into sample: 79 ug recovered of the 90ug spiked (87.8% recovery).

Conclusion: The Silver Citrate PRDM C 5” x 5”/Lot SO 63331 RD product has silver in the form of silver chloride which will still effectively release silver ion but may require a higher amount of silver citrate to provide the antibiotic efficacy in a timely manner. TEST 2:

Antibiotic Effectiveness of Silver Citrate PRDM C 5” x 5 ’’/Lot SO 63331 RD

Procedure:

The Silver Citrate PRDM C 5” x 57Lot SO 63331 RD was placed in a tube with 25 ml_s DI water and solicited for 1 hour. After solication, the membrane was transferred into a beaker and heated to boiling for 30 minutes with constant stirring. The volume was reduced to 10 ml_s and split into 2 x 5 ml_s portions. To one of the tubes an additional 45 ug of silver was added. The two samples were then tested against an inoculum of Staphylococcus Epidermis for 7 days to investigate the reduction in colonies.

Results: Antibiotic Effectiveness

Conclusions: The addition of 90ug silver per Silver Citrate PRDM C 5” x 57Lot SO 63331 RD sheet is the equivalent of 5 x the original content of the product (17.39 ug silver I cm2). Due to the obvious binding of the silver with the chloride in the Silver Citrate PRDM C 5” x 57Lot SO 63331 RD sheet formulation, it was determined to add 5 x the original amount as the silver chloride slowly releases the silver ion and to provide an effective rate of antibiotic, it may be necessary to increase the silver content of the product by this amount. It was seen that the added amount which was an amount 5 x the original amount was effective in reducing the bacteria level by greater than 99.9% whereas in the same time period, the original formulation reduced the level of bacteria by 97.5%.

Summary of Test Results for Antibiotic Efficacy of Preferred Embodiment

Silver ion is a double-edge sword in that it can kill host cells as well as unwanted microbes that interfere with wound healing. The FDA requires sufficient silver to effectively reduce the microbial bioburden to enable efficient healing. Goa I: kill required amount of microbes while doing minimal harm to host tissue.

Original formulation is a single step BioDot Spray operation onto the 3D structure of PermeaDerm procedure:

1 . A silver Citrate/Citric Acid Solution is prepared; a. Dissolve 2.24 grams of USP Citric Acid in 9 ml. of distilled water. b. Add 72.0 mg of Silver Citrate Hydrate and mix to dissolve. c. Add above mixture to ml. volumetric flask and add distilled water to volume.

2. Procedure to make Silver Citrate PermeaDerm Spray solution - 6 microliter of this solution is applied per sq. cm. of the 3D nylon side of Silver PermeaDerm: a. Add 100 ml. of D. I. water to beaker w/ stirring bar and bring temperature to 60 degrees C. b. Gradually add 0.5 mg of MedullaPro (gelatin)until fully dissolved. c. Gradually add 1 .0 mg of lmmuno-10 (Aloe) until fully dissolved. d. Discontinue heating the solution. e. When temperature of solution has cooled about 10 degrees C: gradually add 0.45 grams of Silver Citrate/Citric Acid Solution until fully dissolved. f. Correct for loss of water - same procedure as described in current production “Batch Record Shop Order.” g. Apply 3 microliter per sq. cm. of this solution to the 3D nylon structure to make Silver PermeaDerm.

After Silver PermeaDerm prototypes using the original formulation was manufactured and sterilized with 25 kGy of E Beam the apparent amount of silver present was substantially lower than expected. A concern that the silver might irreversibly be bound to the gelatin or Aloe and unavailable to reduce bacterial bioburden in the wound or in the 3D structure of Silver PermeaDerm.

Preferred formulation for Silver PermeaDerm is a two-step process with following objectives:

1 . A recoverable amount of silver ion from the sterile Silver PermeaDerm biocoat that correlated with the amount of silver applied per sterilization.

2. An amount of silver ion that would effectively reduce the microbial bioburden as required by FDA.

3. An amount of silver ion that minimally harms the host tissue.

The Preferred formulation process:

• The formulation for making Silver PermeaDerm is a two-step biocoat process using the BioDot XYZ spray system. The first step is our Standard method of adding a gelatin/Aloe biocoat (6 microlite per sq. cm. of PermeaDerm) to the 3D surface of PermeaDerm and dried per normal protocol. After the application of the 1 st gelatin/Aloe biocoat and drying (normal protocol) the 2nd biocoat of a silver citrate/citric acid solution (3 microliter per sq. cm. of PermeaDerm) is applied to the 3D surface of PermeaDerm and dried per normal protocol.

• Desire 3 uL (2 ^n< ^ biocoat of Silver Citrate/Citric Acid solution Spray Solution) to dispense 0.54 ug of silver ion or 0.885 ug of silver citrate monohydrate per each square cm surface of PermeaDerm 3D which faces the wound. This concentration is enough to kill 100,000 colony forming units per ml Staphylococcus epidermis at 7, 14 and 21 days. The solution appears stable, has minimal turbidity (as measured at 660 nm) and the silver ion concentration is constant over at least a one month.

• T o make 250 ml_ of Silver Citrate Solution (to deliver 5x the amount (as opposed to formulation on October 27, 2020) of silver ion (0.54 ug per sq. cm in 3 uL) Record weight (+/- 1 %) of:

1 . Beaker plus magnetic stirring bar

2. Sterile water 250 g

3. Silver citrate 74.15 mg

4. USP Citric Acid 56.0 gm

• Procedure (follow standard Trelleborg Batch Record Shop Oder instructions and record keeping) 1 . Add 200 ml_ of H O to beaker w/ stirring bar. Keep constant stirring.

2. Gradually add 56.00 g of citric acid until fully dissolved

3. Gradually add 74.15 mg of silver citrate until fully dissolved

4. When dissolved, add to 250 ml_ volumetric flask and bring to 250 ml_ volume with distilled water.

5. When complete, store at refrigerated temperature (4°C ± 1 °C) for 30 days. After 30 days, filter and weigh filtrate to determine any precipitation. Also, observe solution for cloudiness or any other signs of precipitation of the silver citrate.

6. Apply 5 microliter of this solution to the 3D surface of Standard PermeaDerm to make Silver PermeaDerm

The present invention is a wound closure device that provides an oxygenated, moist but not wet healing environment which by not being wet minimizes bacterial proliferation and optimizes growth of healthy host tissue. The presence of silver ion in the 3D structure of PermeaDerm further enables reduction of harmful bacteria and other microbes.

The new, preferred formulation contained 5X the amount of silver as the original formulation and effectively reduced the bacterial level by 99.9% as compared to the original earlier formulation with 97.9% reduction.

This is the preferred embodiment of the invention. The technology to create this invention is listed as the preferred embodiment of this invention, but other methods are possible and are within the contemplation of this patent.