WO2016176514A1

WO2016176514A1 - Burn, scar, and wound healing aids and bandages

Info

Publication number: WO2016176514A1
Application number: PCT/US2016/029892
Authority: WO
Inventors: Sanjay Dhar; Mohammad Ali KHARAZMI; Mohammad Saeed KHARAZMI
Original assignee: Nugene, Inc.
Priority date: 2015-04-29
Filing date: 2016-04-28
Publication date: 2016-11-03

Abstract

Embodiments include bandages combining sterile dressings with a composition including a conditioned medium. The conditioned medium may be nanoencapsulated or may be dispersed in an emollient base and the applied to the sterile dressing. The conditioned medium may be harvested from culture of human adipocyte-derived stem cells. The nanoencapsulated conditioned medium may be washed and resuspended before applying to the sterile dressing. The emollient base may include defined proportions of oils and emulsifying agents.

Description

TITLE: Burn, scar, and wound healing aids and bandages

CROSS-REFERENCE TO RELATED APPLICATIONS

[001] This application claims priority to US Provisional Application 62/154,120 filed 29 April 2015, the disclosure of which is incorporated by reference.

INVENTORS:

[002] Sanjay Dhar

[003] Mohammad Ali Kharazmi

[004] Mohammad Saeed Kharazmi

APPLICANT:

[005] NuGene, Inc.

BACKGROUND OF THE INVENTION

Field of the Invention

[006] This invention is in the field of dermal care and treatment. In particular, it concerns scar and burn healing aids and bandages that improve the condition of wounds, scars, or burns.

[007] Bandages for wounds or burns are commonly composed of sterile absorbent dressings that are fastened in place by separate fasteners such as tape, adhesives, compressive textiles, or ties. Some bandages may be pretreated with antimicrobials to retard wound infection. For example, wound dressings impregnated with certain healing promoting or microbiocidal materials, such as nanosilver, cause wounds to heal more quickly. Nanosilver is used extensively in wound management, particularly in burn treatment. Other reported applications include chronic wounds, burns injuries in children and neonates, ulcers (including diabetic ulcers, rheumatoid arthritis-associated leg ulcers, and venous ulcers), toxic epidermal necrolysis, healing of donor sites, and meshed skin grafts. Other bandages may be untreated but applied with or over topically applied aids such as antimicrobials, clotting factors, or desiccants.

[008] US2012/0318262 to Lee et al. describe that dressings that may include materials such as pyrvinium or its salts or analogs, gelatin, silver, cellulose, alginate, collagen, a hydrocolloid, a hydrogel, a skin substitute, a wound filler, a growth factor, an antibody, a protease, a protease inhibitor, an antibacterial peptide, an adhesive peptide, a hemostatic agent, living cells, honey, nitric oxide, a corticosteroid, a cytotoxic drug, an antibiotic, an antimicrobial, an antifungal, an antiseptic, nicotine, an anti-platelet drug, an NSAID, colchicine, an anti-coagulant, a vasoconstricting drug or an

immunosuppressive. [009] Many tissues, including living layers of the skin, respond to appropriate mixtures of growth factors to encourage regeneration. For example, US 2012/0065129 Al to Park et al. asserts that a culture medium of adipose-derived stem cells and growth factors isolated from the culture medium can be advantageously applied in drugs, quasi drugs, and cosmetics for wound healing. PCT US2014/034738, commonly assigned with this application, describes dermal treatment compositions including a medium recovered from human adipose-derived stem cell culture (HADSCC).

[0010] US 2011/0091568 to Lipton et al. describes several types of stem cells (including human embryonic stem cells and neural stem cells) and media derived from growth of those cells. Lipton also discloses "plasters, bandages, dressings, gauze pads, patches and the like, containing an appropriate amount of conditioned medium and, optionally, other ingredients. In some cases use may be made of plasters, bandages, dressings, gauze pads, patches and the like which have been impregnated with a topical formulation containing the therapeutic formulation." Upton's dressings serve as a cover for a topical formulation. There is no description of an impregnated dressing suitable for long-term storage prior to use. There is thus a need for dressings that may be prepared ahead of need and stored for days, weeks, or months before application while retaining active healing capability.

[0011] Lipton et al. in particular extols the benefits of embryonic stem cells and neural stem cells, but does not describe HADSCC medium. Nor does Lipton describe "an appropriate amount" other than "optimal percentage of the conditioned media or extract in each composition can vary according to the composition's formulation and the therapeutic effect desired" and by reciting a range of concentrations (apparently in relation to cosmetic procedures) that span more than six orders of magnitude. Lipton et al. describes also does not describe any details which would enable the skilled practitioner to produce effectively the plasters, bandages, dressings, gauze pads, patches and the like. There is thus a need for a bandage that contains the healing=promoting materials present in HADSCC medium in proportions and amounts that support effective healing and that stably contains HADSCC medium for an extended period of time prior to use.

[0012] Lipton' et al. describes incorporation of conditioned medium into liposomes suspended in organic solvent for topical administration. Such compositions may be necessary to maintain liposome stability but may cause deleterious results when applied directly to wounds or burns as the materials suitable for liposome formation are not optimized for contact with damaged tissue. There is thus a need for dressings containing encapsulated active materials without inclusion of organic solvents. [0013] US 8911963 to Epstein et al. describe a therapeutic bandage including conditioned medium and/or processed conditioned medium impregnated into a polymer. The therapeutic bandage is a polymer impregnated with processed conditioned medium that is applied topically in the form of a bandage, patch, or a configured mesh. An embodiment of the bandage is described as a gauze bandage impregnated with a biodegradable polymer comprising conditioned medium. The bandage can be lyophilized and stored for future use, then reconstituted by added moisture or by wound moisture. The polymer is said to permit sustained release of the processed conditioned medium. In other

embodiments, the polymer may be a non-biodegradable polymer that is removed after use. The stem cells can be isolated from adipose tissue. The described therapeutic bandages rely on the polymer embedding to retain and release an effective mix of growth factors from the bandage over an effective time course. HADSCC medium contains a complex mix of growth factors that may not be equally soluble in a given polymer. There is thus a need for a bandage that contains HADSCC medium where retention and release of growth factors does not rely on polymer solubility or embedding.

BRIEF SUMMARY OF THE INVENTION

[0014] In some embodiments, the invention includes a bandage that has a sterile dressing and a composition including a conditioned medium. The conditioned medium may be applied to the sterile dressing. The conditioned medium may be contained in nanocapsules, and may be concentrated by centrifugal filtration prior to loading into the nanocapsules. The nanocapsules may be washed and applied to the sterile dressing as an aqueous suspension and air dried. The nanocapsules may include nanoliposomes or polymer nanocapsules.

[0015] In other embodiments the invention includes a bandage that has a sterile dressing and a composition including a conditioned medium. The conditioned medium may be dispersed in an emollient base and the emollient base applied to the sterile dressing. The conditioned medium may be harvested from culture of human adipocyte-derived stem cells. The conditioned medium may form about 40% by weight of the composition.

[0016] The stem cells may be cultured in Dulbecco's Modified Eagle's Medium or RPM I 1640 medium containing 0.0025 - 0.5% of a nanosilver particulate by weight. The nanosilver particulate includes a stabilizing coating including one or more of citrate, tannic acid, polyvinylpyrrolidone, silica, polyethylene glycol, oligonucleotides, or a peptide. The emollient base may include an oil and an emulsifying agent, with the oil forming about 5-20% by weight of the composition and the emulsifying agent forming about 10-20% of the composition. The oil may include one or more of coconut oil, avocado oil, neem oil, rosemary oil, manuka oil, safflower oil, or geranium oil.

[0017] In some embodiments, the sterile dressing includes a textile or a polymer. In other

embodiments, the sterile dressing is a film, foam, semi-solid gel, pad, gauze, or fabric. The bandage may also include a substance or element for the fixation of the bandage to a wound, such as a tie, an adhesive, a tape, a compression stocking, or an adherent gel. The sterile dressing may have a wound- facing surface with a peripheral area and a central area. The peripheral area may include an adhesive, and the emollient base may be applied to the central area. In other embodiments, the sterile dressing includes a wound-facing surface, and the wound-facing surface includes an adhesive over substantially the entirety of the wound-facing surface. The sterile dressing includes a second surface opposing the wound-facing surface, and the emollient base may applied to the second surface.

[0018] The invention also includes a method including the steps of applying to a wound a bandage that has a sterile dressing and a composition including a conditioned medium. The conditioned medium may be dispersed in an emollient base and the emollient base applied to the sterile dressing. The bandage is then secured to the wound. In some embodiments, the method also includes adding a liquid to the bandage before applying the bandage to the wound. In some embodiments the composition is dried and the liquid rehydrates the dried composition. In other embodiments, the emollient base substantially immobilizes the conditioned medium and the liquid elutes the conditioned medium from the bandage. The wound may be any of a dermal wound, an epidermal wound, a burn, an infectious lesion, a surgical site, an ulcer, or a scar.

[0019] In other embodiments, the invention also includes a bandage having a composition applied to a sterile dressing. The composition has major components of about 5-30% by weight of an oil, about 10- 30% by weight of an emulsifying agent, and about 40-60% by weight of a conditioned medium harvested from culture of human adipocyte-derived stem cells. The composition may also include about 1-5% vitamin E or coconut oil.

[0020] In still other embodiments, the invention includes a method of treating a wound by applying a bandage that has a sterile dressing including a conditioned medium. The conditioned medium may be applied to the sterile dressing. The conditioned medium may be contained in nanocapsules, and may be washed and applied to the sterile dressing as an aqueous suspension and air dried. The method further includes securing the bandage to the wound and may include adding a liquid to the bandage.

BRIEF DESCRIPTION OF THE DRAWING(S) [0021] Not Applicable.

DETAILED DESCRIPTION OF THE INVENTION

[0022] A large number and variety of materials are useful in forming wound dressings. A wound dressing typically includes a film, gel, foam, gauze, textile, polymer, or fabric. Some dressings may include non-absorbable components such as silicone, polyacrylamide gels, conventional textiles, or PTFE. Other dressings, particularly those used for internal wounds, may include polymers that are naturally biodegradable in the body such as fibrin, PLGA, PGA, PLA, polycaprolactone or hyaluronic acid. Dressing may be designed to adhere or to remain free of the wound surface.

[0023] Dressings may be supplemented by creams, ointments, or included materials that promote healing or reduce infection. This invention includes compositions applied to the dressings themselves that promote healing in wounds to which the dressings are applied.

[0024] Appropriate wound closure methods depend on wound severity. Staples or sutures require nurse of physician attendance and sometimes require an anesthetic. Many of these devices leave scars, including scars beyond the wound itself from insertion holes. Varying tensions applied to a laceration or surgical incision between the suturing points cause skin or tissue puckering, increasing the appearance of scars. These skin closure methods may also require follow-up visits to a clinic for removal of sutures or staples.

[0025] Less severe wounds may be closed by over the counter adhesive-based dressings and more complex assemblages of gauze, creams, ointments, tapes, or applied vacuum. Covering a wound with an adhesive bandage may often not be sufficient to close more severe or deeper wounds.

[0026] In the wound healing process, injured tissue is repaired, specialized tissue is regenerated, and new tissue is reorganized. Three major phases are an inflammation stage of zero to three days, a proliferation stage of three to twelve days, and a remodeling phase of a few days to six months or more. In the inflammation phase, platelet aggregation and clotting form a matrix which traps plasma proteins and blood cells and induces the in-migration of various cells from surrounding tissues. In the cellular proliferation phase, new connective or granulation tissue and blood vessels form. In the remodeling phase, granulation tissue is replaced by a network of collagen and elastin fibers producing scar tissue. Each of these phases is at least partly dependent on signaling by growth factors produced by healing and adjacent tissue. The inventive bandages and methods may enhance wound healing during any of these phases. We have found that healing of wounds may be improved when wound dressings include compositions that contain cell culture medium that has been conditioned by growth of human stem cells. In particular, adipose-derived stem cells are derived from connective tissue and vasculature tissue, which may be particularly appropriate to promote healing of skin because of the similarity of the tissue components.

[0027] These conditioned medium dressings supply exogenous growth factors derived from expansion of the stem cells in tissue culture. These exogenous growth factors can augment the response generated by the wound healing process itself.

Stem cell culture medium

[0028] Adipose-derived stem cells are stem cells extracted from adipose tissues. Adipose tissue, like other tissue types, is not a homogenous mixture of a single cell type. Instead adipose tissue includes a combination of fat cells, vasculature, connective tissue, and blood cells. Human adipose tissue is available ex vivo as a result of cosmetic procedures including liposuction. Stem cells may be extracted from such tissue by any of a number of methods known in the art, including treatment with surfactants or enzymes (including proteases such as such as collagenase, or trypsin), maceration, separation by centrifugation, filtering, or settling, ultrasonic treatment, adherent culturing, or some combination of these methods.

[0029] Growth of such adipose-derived stem cells includes supply of nutrients for the cells through provision of an aqueous culture medium. Cells grow in culture in contact with medium and extract nutrients from it. These cells also deliver to the medium products of their growth and metabolism. Among the products are the growth factors and cytokines as well as metabolic products. Conventional tissue culture requires replacement of culture medium as cells use up the nutrients and deliver products that may affect future cell growth. This replacement may be either continuous, with a portion of the medium removed as new medium is added, or intermittent with periodic replacement of some or all of the culture medium in a vessel. Culture medium removed after exposure to cells in culture is known as spent or conditioned medium.

[0030] Culture medium compositions typically include essential amino acids, salts, vitamins, minerals, trace metals, sugars, lipids, and nucleosides. Cell culture medium attempts to supply the components necessary to meet the nutritional needs required to grow cells in a controlled, artificial and in vitro environment. Nutrient formulations, pH, and osmolarity may vary depending on the type of cell cultured, on cell density, and on the culture system employed. The scientific literature includes description of many cell culture medium formulations; a number of such media are commercially available. Conditioned medium contains many of the original components of the medium, as well as a variety of cellular metabolites and secreted proteins, including, for example, biologically active growth factors, inflammatory mediators, and other extracellular proteins. Examples of suitable culture media are Dulbecco's Modified Eagle's Medium and PM I 1640. Such media may be supplemented by other nutrients, growth supporting materials, or antibiotics as is known in the art. An exemplary process of producing a human adipose derived stem cell conditioned ("HADSCC") medium is that described in PCT US2014/034738, commonly assigned with this application, the specification of which is incorporated by reference.

[0031] In some embodiments the bandages of the invention include a HADSCC medium where the media is supplemented with nanosilver particulates before exposure to the human adipose-derived stem cell culture. In these embodiments, the stem cells are cultured in the presence of the nanosilver particulates. Nanosilver can affect the healing of damaged tissue when applied during wound healing or burn healing. Nanosilver may also affect the growth of human adipose-derived stem cells in culture and their production of stem cell products including growth factors, cytokines, stress proteins, and nutrients. Thus HADSCC medium where the stem cells are cultured in the presence of the nanosilver particulates ("HADSCC silver medium") may contain a different mix of stem cell products than HADSCC medium where the stem cells are cultured in the absence of the nanosilver particulates. In some embodiments where the media is supplemented with nanosilver particulates before exposure to the human adipose- derived stem cell culture cells, the media may be otherwise free of or have reduced amounts of antimicrobials such as antibiotics. HADSCC silver medium (other than the presence of nanosilver and possible absence of antibiotics) may be prepared by a similar process to that of HADSCC media without nanosilver. In other embodiments, media harvested from stem cells cultured without nanosilver may be supplemented with nanosilver after harvesting the conditioned media.

[0032] In some embodiments HADSCC medium (or HADSCC silver medium) may be further prepared by concentration or by nanoencapsulation or both. Concentration preparation steps reduce the amount of water in the medium and consequently concentrate the active components of the medium. This step may also serve to desalt the material by passing small ions. Concentration may be performed by any of a variety of methods known in the art, including centrifugal filtration, dialysis, tangential flow filtration, or exposure to water absorbing polymers. In some embodiments, HADSCC medium may be concentrated by centrifugal filtration using a Centricon^* or Microcon^® centrifugal filter devices according to the directions supplied with device by its manufacturer, EMD Millipore of Billerica, Massachusetts. Centricon^® and Microcon^® are registered trademarks of EMD Millipore. Using such a device, active components of the HADSCC medium may concentrated by from about five to about 200 times. We have found particular efficacy for subsequent processing when the active components of the HADSCC medium are concentrated about 50 times. The media treated by this process (or any similar process known in the art) will be referred to as concentrated media, but unless indicated otherwise, subsequent use of the term HADSCC medium includes concentrated media as well as the HADSCC medium as harvested.

HADSCC media from cells cultured without nanosilver may receive nanosilver either before or after concentration.

[0033] HADSCC medium (including concentrated media) may be treated by nanoencapsulation to help enhance stability by protecting the HADSCC medium from exposure to environmental materials or conditions that may degrade the activity of the active materials. Nanoencapsulation may also serve to control the release of active materials to a desired time (e.g. when exposed to a solvent) or at a desired rate. Nanoencapsulation may be performed by any method known in the art, including those reviewed by Subhash Chandra Yadav, Avnesh Kumari, and amdhan Yadav in their paper entitled Development of peptide and protein nanotherapeutics by nanoencapsulation and nanobioconjugation published in Peptides 32 pp. 173-187 (2011). This review is hereby incorporated by reference for its disclosure of methods of nanoencapsulation.

[0034] In some embodiments, a suitable method of nanoencapsulation includes emulsification polymerization using aqueous phase methacrylate monomer and a photoinitator such as benzoin ethyl ether emulsified with HADSCC medium with polyethylene oxide as a stabilizer and exposure to UV light after emulsification to produce poly(methacrylate) encapsulated active components of HADSCC medium. The nanocapsules may range from about 50 to about 1000 nm in diameter. While the nanocapsules may be close to monodiserse (depending on the method of preparation), in some embodiments, the size of nanocapsules may be deliberately widely distributed to control the rate of release of active materials. Widely distributed populations of nanocapsules may be prepared by altering the conditions of emulsification during encapsulation or by mixing two or more batches of nanocapsules with different size.

[0035] In other embodiments, nanoencapsulated HADSCC medium may be prepared as

phospholipid nano-emulsions or nano-liposomes.

[0036] In other embodiments, nanoencapsulated HADSCC medium may be prepared using the apparatus and method described in US patent publication 2008/0182019 entitled Hollow Microsphere Particle Generator. This publication is hereby incorporated by reference for its disclosure of methods of encapsulation of aqueous phase materials.

[0037] Nanoencapsules containing HADSCC medium may be washed by dialysis, by centrifugal filtration, by tangential flow filtration, or by centrifugation and decanting, or by other techniques known in the art, to produce washed nanoencapsulated HADSCC medium. Washing helps remove unreacted monomers or initiator as well as materials not incorporated in nanocapsules. Alternatively, and depending on the materials used in the encapsulation process, nanoencapsulated medium may be used without further processing. HADSCC media from cells cultured without nanosilver may receive nanosilver either before or after nanoencapsulation. After washing, nanoencapsulated media may be resuspended in a buffer, in sterile saline, in water, or in a suspension containing other materials. In some embodiments, nanoencapsulated HADSCC media from cells cultured without nanosilver may be resuspended in buffer or solvent containing nanosilver.

[0038] In some embodiments, nanoencapsulated HADSCC medium may be applied directly to wound dressings and dried in place, as by air drying. The media encapsulated as described above (and by similar processes known in the art) will be referred to as nanoencapsulated media, but unless indicated otherwise, subsequent use of the term HADSCC medium (or media) includes nanoencapsulated media. In other embodiments, otherwise untreated HADSCC medium or concentrated HADSCC medium may be applied to wound dressings and dried in place. In some embodiments these materials may be mixed with other materials such as nanosilver (in the case when the HADSCC media was derived from culture without nanosilver), antimicrobials, antioxidants, or support materials such as thickeners that help to retain the materials in place.

Compositions

[0039] In other embodiments, HADSCC media (including HADSCC silver media), or

nanoencapsulated media may be mixed with other components prior to application to dressings or bandages. These embodiments of the invention may include support ingredients such as oils, vitamins, and emulsifying agents. Oils may include any of a variety of oils helpful to form an emulsion with the aqueous media components. Exemplary oils include coconut oil, avocado oil, neem oil, rosemary oil, manuka oil, safflower oil, and geranium oil. Emulsifying agents may include commercial cold process waxes such as Jeesperse ^® CPW-CG-T (a mixture of cetyl alcohol, sodium acrylate, sodium acryloyl dimethyl taurate copolymer, glyceryl monostearate, and caprylic triglyceride). Jeesperse is a registered trademark of Jeen International Corporation of Fairfield, New Jersey. Other support ingredients may also be present.

[0040] In some embodiments the bandages may include ingredients that make them more suitable for treatment of burns as opposed to other wounds. Bandages more suitable for treatment of burns include vitamin E (Tocopheryl Acetate) in amounts of from about 1-5% by weight of the composition. Bandages more suitable for treatment of wounds may include coconut oil in amounts of from about 1- 5% by weight of the composition.

[0041] We have found that a suitable composition for inclusion in bandages includes about 40% to about 60% by weight of HADSCC media or HADSCC silver media. Oils may range 5-20% with emulsifying agents in the range of 10-20% of the mixture. The balance may be other aqueous ingredients such as aloe vera gel and other plant extracts.

[0042] Without intent to be bound by theory, Applicants believe that the beneficial effects of combining nanosilver particles and HADSSC media may arise because the mix of stem cell products from HADSSC media or from HADSSC silver media complements the nanosilver-activated wound healing response. HADSSC silver media may include especially complementary stem cell products. Further, nanosilver particulates have in some instances been associated with cytotoxic effects. These negative effects of nanosilver may be at least in part ameliorated by the growth factors contained in the HADSSC medium.

Dressings

[0043] Dressings may take many forms, but a dressing is generally a flexible substrate, roughly planar, that conforms to the portion of the body including the wound and protects the wound from the environment. Dressings may hold the edges of wound together, absorb and remove fluids from a wound, add moisture when required, or help to contain and apply medications to the wound. Dressings are generally sterilized to avoid introducing infectious organisms. Any materials added to dressing must be capable of withstanding a sterilization procedure. Sterile dressings suitable for use in embodiments of the invention include textiles, polymers, or composites. Textiles may be woven, such as traditional gauze, or nonwoven sheet or web structures bonded together by entangling fiber or filaments. In other embodiments, the sterile dressing may be a polymer film, foam, semi-solid gel, or pad. Suitable composite materials may include multi-layer assemblages of absorbent, bibulous, or hydrophilic materials covered by protective layers that may either pass or prevent passage of moisture. In some embodiments, the wound-facing surface may include a polymer gel that prevents adherence to the wound. [0044] The bandage may also include a substance or element for fixing of the bandage to a wound, such as a tie, an adhesive, a tape, a compression stocking, or an adherent gel. The sterile dressing may have a wound-facing surface with a peripheral area and a central area. For example, the protective layers may extend beyond the boundary of the absorbent, bibulous, or hydrophilic materials or of a nonadherent wound-facing surface in a peripheral region that includes adhesives to hold the bandage in position. The emollient base containing the HADSCC medium may be applied to the central area.

[0045] In other embodiments, the sterile dressing may include a wound-facing surface that includes an adhesive over substantially the entirety of the wound-facing surface. The sterile dressing includes a second surface opposing the wound-facing surface, and the emollient base may applied to the second surface. This embodiment may be used primarily to bring together edges of a wound, so adhesive may be required over the entire surface.

[0046] The invention also includes a method including the steps of applying to a wound a bandage that has a sterile dressing and a composition including a conditioned medium. The conditioned medium is dispersed in an emollient base and the emollient base is applied to the sterile dressing. The bandage is then secured to the wound. In some embodiments, the method also includes adding a liquid to the bandage before applying the bandage to the wound. In some embodiments the composition is dried and the liquid rehydrates the dried composition. In other embodiments, the emollient base substantially immobilizes the conditioned medium and the liquid elutes the conditioned medium from the bandage. The wound may be any of a dermal wound, an epidermal wound, a burn, an infectious lesion, a surgical site, an ulcer, or a scar.

[0047] Prospective example 1 Nanoencapsulated media applied to bandages

[0048] In some embodiments, nanoencapsulated medium may be applied directly to wound dressings and dried in place, as by air or vacuum-assisted drying. The application process (as well as all processing steps for the HADSCC medium and the subsequent encapsulation and any wash steps take place under sterile conditions (as in a laminar hood or sterile clean room). In some embodiments, component materials or the finished product or packaging may be subject to sterilization steps. Such steps are well known in the art and will not be further discussed.

[0049] Nanoencapsulated media are prepared by harvesting HADSCC medium, concentrating the harvested medium by centrifugal filtration, and nanoencapsulating the concentrated medium. The nanoencapsulated media is subsequently washed, with the nanocapsules resuspended in an application buffer. [0050] Load 17.5 milliliters of freshly-harvested HADSCC medium to a Centricon^® centrifugal filter with Ultracel PL-30 Membrane and spin at 3,500 x g for 20 minutes to produce 0.35 mL of concentrated medium. The concentrated medium is nanoencapsulated by emulsification polymerization. Emulsify the HADSCC medium with twice the volume of 10% aqueous phase methacrylate monomer and 0.5% benzoin ethyl ether as a photoinitator. Expose to UV light after emulsification to produce

poly(methacrylate) nanoencapsulated media. Wash the nanoencapsulated media three times by centrifugation at 3500 x g and aspiration of supernatant. esuspend the washed nanoencapsulated media to a final volume of 1 mL in sterile normal saline to form an application solution.

[0051] Apply the application solution at a coverage of 0.1 mL per cm² of sterile cotton gauze and air dry for 24 hours. The packaged sterile dressing may be stored for up to six months or more prior to use.

[0052] The treated gauze may be packed as a sterile dressing and applied directly to a wound or may first be moistened with sterile water (about O.lmL per cm²).

[0053] Prospective example 2 Composition applied to bandages

[0054] The listed ingredients (with weighed quantities adjusted for batch size) are typically combined in a cleaned and sterilized tank with moderate mixing or in smaller batches in sterile disposable labware. The ingredients may be added in the order listed, one at a time, mixing well between additions. The ingredients may be subject to mechanical emulsification during processing. In some embodiments, the HADSCC media may include nanosilver as discussed above and may include the HADSCC media as a suspension of nanoencapsulated media as described in example 1.

Ingredient % by weight of final mixture

HADSCC Media 40%

Jeesperse^® CPW-CG-T (Cetyl Alcohol/Sodium 10-15%

Acrylate/Sodium Acryloyl Dimethyl Taurate

Copolymer/Glyceryl Monostearate/Caprylic/ Triglyceride)

Butylene Glycol 5-10%

Niacinamide (Vitamin B3) 5-10%

DL-Pantothenic (Vitamin B-5 ) 1-5%

Tetrahexyldecyl Ascorbate (Vitamin C Oily) 1-4%

Glycolic Acid 70% 5-10% Zinc Oxide/Triethoxycaprylylsilane/C12-15 Alkyl 2-5%

Benzoate/DEA Oleth 3 Phosphate

Aloe Vera Gel 1-5%

Avocado Oil 0.1-0.5%

Coconut Oil 1-5%

Manuka Oil 0.1-1.5%

Phenoxyethanol/Sorbic Acid/Caprylyl Glycol 1-3%

[0055] Prospective example 3 Composition applied to bandages for burns

[0056] The listed ingredients (with weighed quantities adjusted for batch size) are typically combined in a cleaned and sterilized tank with moderate mixing or in smaller batches in sterile disposable labware. The ingredients may be added in the order listed, one at a time, mixing well between additions. The ingredients may be subject to mechanical emulsification during processing. In some embodiments, the HADSCC media may include nanosilver as discussed above and may include the HADSCC media as a suspension of nanoencapsulated media as described in example 1.

Ingredient % by weight of final mixture

HADSCC Media 40%

Jeesperse^® CPW-CG-T (Cetyl Alcohol/Sodium 10-15%

Acrylate/Sodium Acryloyl Dimethyl Taurate

Copolymer/Glyceryl Monostearate/Caprylic/ Triglyceride)

Butylene Glycol 5-10%

Niacinamide (Vitamin B3) 5-10%

DL-Pantothenic (Vitamin B-5 ) 1-5%

Tocopheryl Acetate (Vitamin E) 1-5%

Tetrahexyldecyl Ascorbate (Vitamin C Oily) 1- 4%

Glycolic Acid 70% 5-10%

Zinc Oxide/Triethoxycaprylylsilane/C12-15 Alkyl 2- 5% Benzoate/DEA Oleth 3 Phosphate

Avocado Oil

Manuka Oil

Phenoxyethanol/Sorbic Acid/Caprylyl Glyi Safflower Oil Geranium Oil

[0057] This specification discloses various aspects of the invention with reference to particular embodiments, but it should be understood that any of the features, functions, materials, or

characteristics may be combined with any other of the described features, functions, materials, or characteristics. The description of particular features, functions, materials, or characteristics in connection with a particular embodiment is exemplary only; it should be understood that it is within the knowledge of one skilled in the art to include such feature, structure, or characteristic in connection with other embodiments whether or not explicitly described. We intend the scope of the appended claims to encompass such alternative embodiments. Variations on these described embodiments will become apparent to those of ordinary skill in the art upon reading the description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than specifically described herein. Accordingly, this specification and claims include all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law.

[0058] Unless otherwise indicated, all numbers used in the specification and claims are to be understood as being modified in all instances by the term "about." Unless indicated to the contrary, the numerical values in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained.

[0059] The terms "a," "an," "the" and similar referents used in the context of describing the invention (especially in the context of the following claims) are intended to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the claims. No language in the specification should be construed as indicating any non- claimed element essential to the practice of the invention.

[0060] This document incorporates disclosure from other documents by reference. In the event of any conflicts between the referenced disclosure and the express disclosure of this document, the disclosure of this document shall control.

Claims

CLAIMS We claim:

1. A bandage comprising:

a sterile dressing; and

a conditioned medium applied to the sterile dressing,

wherein the conditioned medium is contained in nanocapsules.

2. The bandage of claim 1, wherein the conditioned medium is concentrated by centrifugal filtration.

3. The bandage of claim 1 or 2, wherein the nanocapsules are applied to the sterile dressing as an aqueous suspension and air dried.

4. The bandage of claim 1, wherein the nanocapsules include nanoliposomes.

5. The bandage of claim 1, wherein the nanocapsules include washed polymer nanocapsules.

6. The bandage of claim 3, wherein the aqueous suspension includes nanosilver.

7. A bandage comprising:

a sterile dressing; and

a composition including a conditioned medium,

wherein the conditioned medium is dispersed in an emollient base, and the emollient base is applied to the sterile dressing.

8. The bandage of claim 7, wherein the conditioned medium is harvested from culture of human

adipocyte-derived stem cells.

9. The bandage of claim 8, wherein the conditioned medium forms about 40% by weight of the

composition.

10. The bandage of claim 9, wherein the stem cells are cultured in one of Dulbecco's Modified Eagle's Medium or PM I 1640 medium containing 0.0025 - 0.5% of a nanosilver particulate by weight of the medium.

11. The bandage of claim 10, wherein the nanosilver particulate includes a stabilizing coating including one or more of citrate, tannic acid, polyvinylpyrrolidone, silica, polyethylene glycol, oligonucleotides, or a peptide.

12. The bandage of claim 11, wherein the emollient base includes an oil and an emulsifying agent.

13. The bandage of claim 12, wherein the oil forms about 5-20% by weight of the composition and the emulsifying agent forms about 10-20% of the composition.

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14. The bandage of claim 13, wherein the oil includes one or more of coconut oil, avocado oil, neem oil, rosemary oil, manuka oil, safflower oil, or geranium oil.

15. The bandage of claim 14, wherein the sterile dressing includes a textile or a polymer.

16. The bandage of claim 14, wherein the sterile dressing includes one or more of a film, a foam, a semisolid gel, a pad, a gauze, or a fabric.

17. The bandage of claim 16, wherein the sterile dressing includes a second surface opposing the

wound-facing surface, and wherein the emollient base is applied to the second surface.

18. A method of treating a wound comprising:

applying the bandage of claim 1 to the wound;

securing the bandage to the wound.

19. The method of claim 18, wherein the nanocapsules are washed and wherein the bandage is air dried, the method further comprising adding a liquid to the bandage.

20. A bandage comprising:

a sterile dressing; and

a composition consisting essentially of:

about 5-30% by weight of an oil;

about 10-30% by weight of an emulsifying agent;

about 40-60% by weight of a conditioned medium harvested from culture of human adipocyte-derived stem cells.

21. The bandage composition of claim 20, further comprising about 1-5% vitamin E or coconut oil.

22. A bandage comprising:

a sterile dressing; and

a conditioned medium applied to the sterile dressing,

wherein the conditioned medium is concentrated from media harvested from culture of human adipose derived stem cells, and wherein the conditioned media contains nanosilver.

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