Rhamnolipids for biofilm control during wound closure

Abstract

The technology presented herein, in general, relates to the use of biosurfactants, such as rhamnolipids, for the treating wounds. More particularly, the present technology relates to a method for reducing or inhibiting the development of biofilm during to promote wound closure, by applying a composition comprising a mixture of specific rhamnolipids to the wound.

Description

RHAMNOLIPIDS FOR BIOFILM CONTROL DURING WOUND CLOSURECROSS-REFERENCE TO RELATED APPLICATIONSThis application claims priority to United States Provisional Application No. 63 / 724979, filed November 26, 2024. The entire specification of this provisional application is hereby incorporated by reference.FIELD OF THE INVENTION

[0001] The present technology, in general, relates to the use of biosurfactants, such as rhamnolipids, for controlling, treating, and / or otherwise removing biofilm during wound healing and / or closure. More particularly, the present technology relates to a method for treating wounds, by applying a composition comprising a mixture of specific rhamnolipids to the wound, wherein application of the composition controls, treats, and / or otherwise removes biofilm.BACKGROUND OF THE INVENTION

[0002] The wound-healing process consists of a progression through a number of highly integrated and overlapping phases, including: hemostasis (e.g. vascular constriction, platelet aggregation, degranulation, and fibrin formation (thrombus)); inflammation (e.g., neutrophil infiltration, monocyte infiltration and differentiation to macrophage, and lymphocyte infiltration); proliferation (e.g., re-epithelialization, angiogenesis, collagen synthesis, and ECM formation); and tissue remodeling or resolution (e.g., collagen remodeling, and vascular maturation and regression).

[0003] These phases, and their associated biophysiological functions, must occur in the proper sequence, at specific times and durations. Optimal wound healing in adult humans generally involves at least the following the events: (1 ) rapid hemostasis; (2) appropriate inflammation; (3) mesenchymal cell differentiation, proliferation, and migration to the wound site; (4) suitable angiogenesis; (5) prompt re-epithelialization (regrowth of epithelial tissue over the wound surface); and (6) proper synthesis, crosslinking, and alignment of collagen to provide strength to the healing tissue.

[0004] Wounds that exhibit both normal and impaired healing, including both acute and chronic wounds, generally have failed to progress through the normal stages of healing (i.e., enter a state of pathologic inflammation due to a postponed, incomplete, or uncoordinated healing process). Biofilms are known to stall the healing process in the inflammatory phase.

[0005] There is therefore an ongoing need for new wound healing compositions, and methods of use, that facilitate both a robust and coordinated healing process, including compositions and methods that facilitate the removal of biofilm to promote wound closure.

[0006] There has also been a recent trend to formulate products with ingredients that are based on renewable raw materials. Such ingredients are considered “green” or “natural”, since they are derived from renewable and / or sustainable sources. As a result, they are more environmentally friendly than ingredients derived from fossil fuels or other non-renewable sources. An ingredient having a high Bio-renewable Carbon Index (BCI), such as greater than 80, indicates that the ingredient contains carbons that are derived primarily from plant, animal or marine-based sources.

[0007] Rhamnolipids are interface-active glycolipids produced by various bacterial species and are an example of a “green” ingredient, since they can be prepared by means of fermentation based on renewable raw materials. It would be desirable to provide compositions that include active ingredients derived from renewable sources, such as rhamnolipids, that can be used to facilitate the treatment and healing of wounds. Providing wound healing / treatment compositions comprising rhamnolipids would satisfy sustainability goals of ensuring sustainable consumption through the use of bio-based antibacterial materials.

[0008] Applicants have determined that mixtures of rhamnolipid salts can meet the above objectives while also advancing UN Sustainability Goals (“SDG”). The rhamnolipid salt mixtures of the present technology contribute to better health and well-being by delivering equal or better efficacy in the treatment of wounds. The rhamnolipid salt mixtures are advantageously bio-based, renewably sourced actives obtained from a bacterial fermentation process that generates biodegradable waste products that are less impactful on the environment. These benefits further SDG #3 (Good Health and Wellbeing) and SDG #12 (Responsible Consumption and Production).SUMMARY OF THE INVENTION

[0009] One aspect of the present technology is directed to compositions and methods for the improved inhibition and / or removal of pathogenic biofilms (e.g., prevention, control or elimination of infection). The methods of the present disclosure comprise applying a composition to a wound, wherein the composition comprises at least one rhamnolipid, and wherein application of the composition improves the inhibition and / or removal ofpathogenic biofilms during healing and closure of the wound. In some embodiments, the wound being treated is an acute wound, including an incision, a laceration, an abrasion, an avulsion, a puncture, a penetration, or a burn wound. Some embodiments include treating chronic wounds (e.g., diabetic foot ulcers (DFU), pressure ulcers (PU) or venous leg ulcers (VLU)). In other embodiments, the composition applied to the wound comprises mono-rhamnolipid, di-rhamnolipid, or a combination of both mono- and di-rhamnolipids. In further embodiments, in response to the application of the rhamnolipid compositions presented herein the rate or time to wound closure is accelerated, together with the prevention, treatment, or removal of associated pathogenic biofilms.

[0010] In another aspect, the present technology is directed to a rhamnolipid composition for use in methods of reducing the formation and / or viability of biofilms associated with the treatment of wounds, wherein the composition comprises a mixture of mono-rhamnolipids and di-rhamnolipids having a mono-rhamnolipids:di-rhamnolipids weight ratio of about 40:60 to about 60:40, preferably about 40:60 to about 48:52. In addition, the rhamnolipid composition comprises, based on the total weight of rhamnolipids present in the composition: an amount of 010-010 mono-rhamnolipid of about 29% to about 40% by weight, preferably 29% to about 37% by weight; an amount of C10-C10 di-rhamnolipid of about 35% to about 50% by weight, preferably about 35% to about 45%; an amount of 08-010 mono-rhamnolipid of about 2% to about 5% by weight; an amount of 08-010 di-rhamnolipid of about 2% to about 5% by weight; an amount of 08-010 di-rhamnolipid of about 2% to about 5% by weight; an amount of 010- 012 mono-rhamnolipid of about 2% to about 6% by weight; and an amount of 010-012 di-rhamnolipid of about 8% to about 14% by weight; an amount of 010-012:1 di-rhamnolipid of about 2% to about 5% by weight; an amount of 012-012 di-rhamnolipid of about 0.2% to about 0.4% by weight. The composition further comprises at least one acceptable carrier, and optionally one or more additives, in an amount to total 100% by weight of the composition.

[0011] In yet another aspect, the present technology is directed to a rhamnolipid composition for use in methods of reducing the formation and / or viability of biofilms associated with the treatment of wounds, wherein the mixture of rhamnolipids is in an amount of about 0.1 % to about 10%, based on the total weight of the composition.

[0012] In a further aspect, the present technology is directed to a rhamnolipid composition for use in methods of reducing the formation and / or viability of biofilms associated with the treatment of wounds, wherein the composition has a mixture of rhamnolipids and at least one acceptable carrier, and optionally one or more additives. In this aspect, the at least one acceptable carrier can be water alone or in combination with an alcohol (e.g., ethanol, isopropanol, or benzyl alcohol) or glycol (e.g., propylene glycol or polyethylene glycol).

[0013] In another aspect, the present technology is directed to a rhamnolipid composition for use in methods of reducing the formation and / or viability of biofilms associated with the treatment of wounds, wherein the method comprises topical administration of the composition. In this aspect, the composition can be formulated as, for example, a lotion, cream, gel, paste, ointment, transdermal patch, or powder.

[0014] In a still further aspect, the present technology is directed to methods of inhibiting biofilm formation (or removing established biofilms) to promote wound healing, the methods including the application of any one of the compositions disclosed herein.

[0015] In yet another aspect, there are provided rhamnolipid containing compositions which when applied to a wound, in accordance to methods disclosed herein, can reduce the level of a multidrug resistant organisms (MDRO) by as much as a 5-log reduction (e.g., from 109colony forming units (CFU) per wound to <104CFU per wound), within 1 , 2, 3, or 4 weeks of treatment. In this aspect, the MDRO is MRSA (methicillin-resistant Staphylococcus aureus), VRE (Vancomycin-Resistant Enterococci), or another common wound pathogen (e.g., Pseudomonas aeruginosa Streptococcus pyogenes, Enterobacter cloacae, Escherichia coli, Corynebacteria, Proteus mirabilis, Proteus vulgaris, and Morganella morganii).

[0016] In a further aspect, there are provided rhamnolipid containing compositions which when applied to a wound, in accordance to methods disclosed herein, disrupts the extracellular polymeric substance (EPS) to destabilize or remove the biofilm. In this aspect, the EPS component removed can be, for example, either a microbe, a protein, a lipid, a polysaccharide, an extracellular DNA (eDNA), or combination thereof.BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 shows the quantitative image analysis of an S. Aureus Infection, determined using MolecuLight i:x™ device as set forth herein.

[0018] Figure 2 shows the Calgary Biofilm Device plate set up, in accordance with Example 2.

[0019] Figure 3 shows the Calgary Film Device (Innovotech: 19171 ), with pegs sitting inside the wells.

[0020] Figure 4 graphically shows the Log Reduction of MRSA viable cells using the Prevention and Dissociation methods set forth in Example 2.

[0021] Figure 5 graphically shows the Log Reduction of Methicillin resistantStaphylococcus aureus viable cells using the Prevention method set forth in Example 2.

[0022] Figure 6 graphically shows the Log Reduction of Methicillin resistantStaphylococcus aureus viable cells using the Dissociation method set forth in Example 2.

[0023] Figure 7 graphically shows the Log Reduction of P. aeruginosa viable cells using the Prevention and Dissociation method set forth in Example 2.

[0024] Figure 8 graphically shows the Log Reduction of P. aeruginosa viable cells after 24 hours using the Dissociation method set forth in Example 2.

[0025] Figure 9 graphically shows the Log Reduction of P. aeruginosa viable cells after 24 hours using the Prevention method set forth in Example 2.DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] While the present technology will be described in connection with one or more preferred embodiments, it will be understood by those skilled in the art that the technology is not limited to only those specific embodiments. To the contrary, the presently described technology includes all alternatives, modifications, and equivalents as may be included within the spirit and scope of the appended claims.

[0027] Generally, the present disclosure is directed to compositions and methods for treating a wound, including the elimination of polymicrobial biofilms (e.g., S. aureus, P. aeruginosa, C. albicans, E. coli, S. pyogenes, and E. faecal is). The methods disclosedherein comprise applying a composition to a wound, wherein the composition comprises at least one rhamnolipid, and wherein application of the composition to the wound improves the inhibition and / or removal of pathogenic biofilms. Wounds treated herein include, for example, acute wounds including incisions, lacerations, abrasions, avulsions, punctures, penetrations, or burns.

[0028] Biofilms in chronic wounds tend to be polymicrobial and consist of at least 2-3 microbe species. Mature biofilms are a dense and heterogeneous extracellular polymeric substance (EPS) composed of living, senescent, dead microbes and polysaccharides, proteins, lipids and extracellular DNA. The EPS makes the microbes more resistant to mechanical sheer, immune evasion and tolerance to antibiotics. Many of these microbes found in chronic and acute wounds are multidrug resistant such as methicillin- resistant Staphylococcus aureus (MRSA . In a biofilm Pseudomonas aeruginosa can evade antibiotics through efflux pumps, and antibiotic inactivating enzymes, while Enterococcus faecalis is commonly found to be resistant to vancomycin (VRE). Several of the other common wound pathogens (Streptococcus pyogenes, Enterobacter cloacae, Escherichia coli, Corynebacteria, Proteus mirabilis, Proteus vulgaris, and Morganella morganii) are resistant to many different types of antibiotics, including aminopenicillins, cephalosporins, fluoroquinolones, sulfonamides, aminoglycosides, beta-lactam-beta- lactamase-inhibitors, and carbapenems.

[0029] Since systemic antibiotics are often ineffective at treating these multidrug resistant organisms (MDRO) in a chronic or acute wound novel approaches are required to remove or destabilize the polymicrobial wound biofilms. See, e.g., the following: (1 ) Gjodsbol K, Christensen JJ, Karlsmark T, Jorgensen B, Klein BM, Krogfelt KA. Multiplebacterial species reside in chronic wounds: a longitudinal study. Int Wound J. 2006 Sep;3(3):225-31 . doi: 10.1 1 11 / j.1742-481 X.2006.00159.x. PMID: 16984578; PMCID: PMC7951738; (2) Rather MA, Gupta K, Mandal M. Microbial biofilm: formation, architecture, antibiotic resistance, and control strategies. Braz J Microbiol. 2021 Dec;52(4):1701 -1718. doi: 10.1007 / s42770-021 -00624-x. Epub 2021 Sep 23. PMID: 34558029; PMCID: PMC8578483; and (3) Bowler PG, Welsby S, Towers V, Booth R, Hogarth A, Rowlands V, Joseph A, Jones SA. Multidrug-resistant organisms, wounds and topical antimicrobial protection. Int Wound J. 2012 Aug;9(4):387-96. doi: 10.1 111 / j.1742- 481 X.2012.00991 .x. Epub 2012 May 29. PMID: 22640181 ; PMCID: PMC7950580. These references are herein incorporated by reference.

[0030] As defined herein, a “rhamnolipid” is a glycolipid that has a lipid portion that includes one or more, typically linear, saturated or unsaturated B-hydroxy-carboxylic acid moieties and a saccharide portion of one or two units of rhamnose.

[0031] The saccharide portion and the lipid portion are linked via a B-glycosidic bond between the 1 -OH group of a rhamnose moiety of the saccharide portion and the 3-OH group of a B-hydroxy-carboxylic acid of the lipid portion. Thus, the carboxylic acid of one carboxylic acid moiety defines the end of the rhamnolipid. Where more than one rhamnose-moiety is included in a rhamnolipid, each of the rhamnose moieties not linked to the lipid portion is linked to another rhamnose moiety via a 1 ,4B-glycosidic bond. In embodiments where two or more B-hydroxy-carboxylic acids are present in a rhamnolipid, the B-hydroxy-carboxylic acid moieties are selected independently from each other. B- hydroxy carboxylic acid moieties may in some embodiments be identical. In some embodiments, they are different from each other.

[0032] The present technology generally relates to a wound care / treatment composition that comprises a particular mixture of rhamnolipids in their salt form. The rhamnolipids may have the following structure (I):In this formula, R9is a hydrogen atom (H) or an aliphatic group that has a main chain of one to about 46, such as one to about 42, one to about 40, one to about 38, one to about 36, one to about 34, one to about 30, one to about 28, including e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27 or 28 carbon atoms and one to about three, including two, oxygen atoms. In some embodiments, the main chain of the respective aliphatic group carries a terminal carboxylic acid group and / or an internal ester group. As an illustrative example in this regard, R9may be of the formula - CH(R5)— -CHz- COOR6. In these illustrative moieties, R5may be an aliphatic moiety with a main chain that has a length from 1 to about 19, such as from 1 to about 17, from 1 to about 15, from 1 to about 13, about 2 to about 13, about 3 to about 13 or about 4 to about 13, including e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 or 12 carbon atoms. R4in formula (I) is a hydrogen atom (H), or a rhamnopyranosyl moiety. R6is a hydrogen atom.

[0033] The term "aliphatic" means, unless otherwise stated, a straight or branched hydrocarbon chain, which may be saturated or mono- or poly-unsaturated and include heteroatoms. The term "heteroatom" as used herein means an atom of any element other than carbon or hydrogen. Herein, an unsaturated aliphatic group contains one or more double bonds (alkenyl moieties). The branches of the hydrocarbon chain may include linear chains as well as non-aromatic cyclic elements. The hydrocarbon chain, which may, unless otherwise stated, be of any length, and contain any number of branches. Typically, the hydrocarbon (main) chain includes 1 to about 5, to about 10, to about 15 or to about 20 carbon atoms. Examples of alkenyl moieties are straight-chain or branched hydrocarbon moieties that contain one or more double bonds. Alkenyl moieties generally contain about two to about twenty carbon atoms and one or more, for instance two, double bonds, such as about two to about ten carbon atoms, and one double bond. Examples of alkyl groups are methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, the n isomers of these radicals, isopropyl, isobutyl, isopentyl, sec-butyl, tert-butyl, neopentyl, 3,3-dimethylbutyl. Both the main chain as well as the branches may furthermore contain heteroatoms as for instance N, O, S, Se or Si or a carbon atom may be replaced by one of these heteroatoms. An aliphatic moiety may be substituted or unsubstituted with one or more functional groups. Substituents may be any functional group, as for example, but not limited to, amino, amido, carbonyl, carboxyl, hydroxyl, nitro, thiol and sulfonyl.

[0034] In a more particular embodiment, the rhamnolipid salts in said structure have the structure (II):wherein x is 1 or 2, y is 4, 6 or 8, z is 4, 6, or 8, and M is H, or a metal, such as alkali metals Li, Na, or K, alkali earth metals Mg or Ca, or transition metals Mn, Fe, Cu, Ag, or Zn. In the cases of the alkali earth and transition metals, multiple rhamnolipid salt moieties may associate with each metal.

[0035] The mixture of rhamnolipids preferably comprises mono (where x=1 ) and di (where x=2) rhamnolipids where y and z are 6 and M is H or Na. The mono-rhamnolipid may be referred to as Rha-C10-C10, with a formula of C26H48O9. The IUPAC Name is 3- [3-[(2R,3R,4R,5R,6S)-3,4,5-trihydroxy-6-methyloxan-2-yl]oxydecanoyloxy]decanoic acid. The di-rhamnolipid may be referred to as RhaRha-C10-C10, with a formula of C32H58O13. The IUPAC name is 3-[3-[ 4, 5-dihydroxy-6-methyl-3-(3,4,5-trihydroxy-6- methyloxan-2-yl)oxyoxan-2-yl]oxydecanoyloxy]decanoic acid. In general, the mixture of rhamnolipids disclosed herein comprises various types of mono and di rhamnolipids and the mixture specifically encompasses all possible combinations of mono and di rhamnolipids as disclosed herein. Further, unless otherwise stated, an amount of an individual mono or di-rhamnolipid as disclosed herein means that the respective mono ordi-rhamnolipid can be present in the mixture of rhamnolipids in the indicated amount, and the mixture of rhamnolipids disclosed herein specifically includes all possible combinations of amounts of mono and di-rhamnolipids as disclosed herein. Generally preferred mixtures of rhamnolipids are SEP-RM rhamnolipid compositions as described below.

[0036] Rha-C10-C10 may be present in the mixture in an amount of about 29% to about 40%, alternatively about 30% to about 40%, alternatively about 29% to about 37%, alternatively about 35% to about 37% by weight based on the total weight of rhamnolipids. RhaRha-C10-C10 may be present in the mixture in an amount of about 35% to about 50%, alternatively about 35% to about 45%, alternatively about 36% to about 40%, alternatively about 36% to about 38% by weight based on the total weight of rhamnolipids.

[0037] In addition to Rha-C10-C10 and RhaRha-C10-C10, the mixture of rhamnolipids may comprise RhaRha-C10-C12 in an amount of about 8% to about 14%, alternatively about 9% to about 12%, alternatively about 10% to about 12.5% by weight based on the total weight of rhamnolipids, and Rha-C10-C12 in an amount of about 2% to about 6% by weight, alternatively about 2% to about 5%, alternatively about 3.5% to about 5% by weight based on the total weight of rhamnolipids. The mixture of rhamnolipids may also comprise RhaRha-C10-C12:1 in an amount of about 2% to about 5% by weight, alternatively about 3% to about 5% by weight, based on the total weight of rhamnolipids, an amount of RhaRha-C8-C10 in the range of about 2% to about 5% by weight, alternatively about 2% to about 4% by weight, based on the total weight of rhamnolipids, and an amount of Rha-C8-C10 in the range of about 2% to about 5% by weight, alternatively about 2% to about 4% by weight, based on the total weight of rhamnolipids.The mixture of rhamnolipids may also comprise Rha-Rha 012-012 in an amount of about 0.1 % to about 0.5% by weight, alternatively about 0.2% to about 0.4% by weight, alternatively about 0.2% to about 0.3% by weight, alternatively about 0.25% to about 0.3% by weight, based on the total weight of rhamnolipids present in the composition.

[0038] The mixture of rhamnolipids may comprise a mixture of mono-rhamnolipids and di-rhamnolipids. The mono-rhamnolipids may be present in an amount of about 40% to about 50%, preferably about 42% to about 48%, based on the total weight of rhamnolipids in the mixture. The di-rhamnolipids may be present in an amount of about 50% to about 60% by weight, preferably about 52% to about 58%, based on the total weight of rhamnolipids. The ratio of mono-rhamnolipids:di-rhamnolipids can be from about 40:60 to about 60:40, alternatively about 40:60 to about 50:50, alternatively about 40:60 to about 48:52, preferably about 42:58 to about 48:52. In some embodiments, the mono- rhamnolipids may be present in an amount of about 10% to about 90%, alternatively about 15% to about 90%, alternatively about 20% to about 90%, alternatively about 30% to about 90%, alternatively about 40% to about 90%, alternatively about 10% to about 80%, alternatively about 20% to about 80%, alternatively about 30% to about 80%, alternatively about 40% to about 80%, alternatively about 10% to about 70%, alternatively about 15% to about 70%, alternatively about 20% to about 70%, alternatively about 30% to about 70% by weight, based on the total weight of rhamnolipids in the mixture and as measured by high-performance liquid chromatography (HPLC).

[0039] In some embodiments, the mono-rhamnolipids may be present in an amount of about 10% to about 60%, alternatively about 15% to about 60%, alternatively about 20% to about 60%, alternatively about 25% to about 60%, alternatively about 30% to about60%, alternatively about 25% to about 50%, alternatively about 10% to about 48%, alternatively about 15% to about 48%, alternatively about 20% to about 48%, alternatively about 25% to about 48%, alternatively about 30% to about 48% by weight, based on the total weight of rhamnolipids in the mixture, as determined by HPLC.

[0040] In some embodiments, the di-rhamnolipids may be present in an amount of about 10% to about 90%, alternatively about 15% to about 90%, alternatively about 20% to about 90%, alternatively about 30% to about 90%, alternatively about 40% to about 90%, alternatively about 50% to about 90%, alternatively about 10% to about 80%, alternatively about 20% to about 80%, alternatively about 30% to about 80%, alternatively about 40% to about 80%, alternatively about 50% to about 80%, alternatively about 10% to about 75%, alternatively about 15% to about 75%, alternatively about 20% to about 75%, alternatively about 30% to about 75%, alternatively about 40% to about 75%, alternatively about 50% to about 75% by weight, based on the total weight of rhamnolipids in the mixture, as determined by HPLC.

[0041] In some embodiments, the di-rhamnolipids may be present in an amount of about 40% to about 70%, alternatively about 50% to about 70%, alternatively about 52% to about 90%, alternatively about 52% to about 85%, alternatively about 52% to about 80%, alternatively about 52% to about 75%, alternatively about 52% to about 70%, based on the total weight of rhamnolipids in the mixture, as determined by HPLC.

[0042] The ratio of mono-rhamnolipids:di-rhamnolipids can be from about 10:90 to about 90:10, alternatively 10:90 to about 60:40, alternatively about 15:85 to about 60:40, alternatively about 20:80 to about 60:40, alternatively about 25:75 to about 60:40, alternatively about 30:70 to about 60:40, alternatively about 10:90 to about 48:52,alternatively about 15:85 to about 48:52, alternatively about 20:80 to about 48:52, alternatively about 25:75 to about 48:52, alternatively about 30:70 to about 48:52. In preferred embodiments, the ratio of mono-rhamnolipids:di-rhamnolipids is about 25:75 to about 60:40, alternatively about 25:75 to about 50:50, or about 25:72 to about 48:52.

[0043] The mixture of rhamnolipids preferably comprises mono (where x=1 ) and di (where x=2) rhamnolipids where y and z are 6 and M is H or Na. The mono-rhamnolipid may be referred to as Rha-C10-C10, with a formula of C26H48O9. The IUPAC Name is 3-[3-[(2R,3R,4R,5R,6S)-3,4,5-trihydroxy-6-methyloxan-2-yl]oxydecanoyloxy]decanoic acid. The di-rhamnolipid may be referred to as RhaRha-C10-C10, with a formula of C32H58O13. The IUPAC name is 3-[3-[ 4, 5-dihydroxy-6-methyl-3-(3,4,5-trihydroxy-6- methyloxan-2-yl)oxyoxan-2-yl]oxydecanoyloxy]decanoic acid.

[0044] Rha-C10-C10 may be present in the mixture in an amount of about 5% to about 85%, alternatively about 5% to about 50%, alternatively about 5% to about 38%, alternatively about 10% to about 38%, alternatively about 15% to about 38%, alternatively about 20% to about 38%, alternatively about 25% to about 37.5%, alternatively about 29% to about 37.5%, by weight based on the total weight of rhamnolipids. RhaRha-C10- C10 may be present in the mixture in an amount of about 5% to about 85%, alternatively about 10% to about 80%, alternatively about 20% to about 80%, alternatively about 30% to about 80%, alternatively about 35% to about 75%, alternatively about 35% to about 65%, alternatively about 35% to about 60%, alternatively about 35% to about 55%, alternatively about 35.5% to about 50%, alternatively about 36% to about 45%, alternatively about 40% to about 60% by weight based on the total weight of rhamnolipids.

[0045] In addition to Rha-010-010 and RhaRha-010-010, the mixture of rhamnolipids may comprise RhaRha-C10-C12 in an amount of about 3% to about 15%, alternatively about 3% to about 12%, alternatively about 3% to about 12.5% by weight based on the total weight of rhamnolipids, and Rha-010-C12 in an amount of about 0.2% to about 6% by weight, alternatively about 0.4% to about 5% by weight based on the total weight of rhamnolipids. The mixture of rhamnolipids may also comprise RhaRha-C10-C12:1 in an amount of about 0.04% to about 4% by weight, alternatively about 0.05% to about 4% by weight, alternatively about 0.05% to about 3% by weight, based on the total weight of rhamnolipids, an amount of RhaRha-C8-C10 in the range of about 0.2% to about 5% by weight, alternatively about 0.6% to about 4% by weight, based on the total weight of rhamnolipids, and an amount of Rha-C8-C10 in the range of about 0.2% to about 5% by weight, alternatively about 0.6% to about 4% by weight, based on the total weight of rhamnolipids. In some embodiments, the mixture of rhamnolipids may also comprise an amount of RhaRha-012-C12 in the range of about 3% to about 10% by weight, based on the total weight of rhamnolipids in the mixture.

[0046] In some embodiments, the mixture of rhamnolipids comprises a mixture of mono-rhamnolipids and di-rhamnolipids in a weight ratio of mono-rhamnolipids:di- rhamnolipids in the range of about 25:75 to about 60:40, alternatively about 25:75 to about 50:50, or alternatively about 25:75 to about 45:55.

[0047] The mono-rhamnolipid may comprise one or more mono-rhamnolipid-mono- lipidic congeners, including for example: Rha-C8-:2; Rha-C8; Rha-C10; Rha-C12:2; Rha- C12; Rha-014:2; or combinations thereof. The mono-rhamnolipid may also comprise one or more mono-rhamnolipid-di-lipidic congeners, including for example: Rha-C8-C8; Rha-C8-C10:1 ; Rha-C10:1 -C8; Rha-C8-C10; Rha-C10-C8; Rha-C10-C10:1 ; Rha-010-010; Rha-C8-C12; Rha-C12-C8; Rha-C10-C12:1 ; Rha-C12:1 -C10; Rha-C10-12; Rha-C12- C10; Rha-C10-014:1 ; Rha-C12-C12:1 ; Rha-C10-C14; Rha-C12-C12; Rha-C12-C14; Rha-C14-C14; Rha-C14-C16; Rha-C16-C16; Rha-C10-C10-CH3; Decenoyl-Rha-C10- 010; or combinations thereof.

[0048] The di-rhamnolipid may comprise one or more di-rhamnolipid-mono-lipidic congeners, including for example: Rha-Rha-C8; Rha-Rha-C10; Rha-Rha-C12:1 ; Rha- Rha-C12; Rha-Rha-C14; or combinations thereof. The di-rhamnolipid may also comprise one or more di-rhamnolipid-di-lipidic congeners, including for example: Rha-Rha-C8-C8; Rha-Rha-C8-C10; Rha-Rha-C10-C8; Rha-Rha-C10-010:1 ; Rha-Rha-C10-C10; Rha- Rha-C8-C12:1 ; Rha-Rha-C12:1 -08; Rha-Rha-C10-012:1 ; Rha-Rha-C12:1 -C10; Rha- Rha-C10-C12; Rha-Rha-C12-010; Rha-Rha-C10-C14:1 ; Rha-Rha-C12-C12:1 ; Rha- Rha-C12:1 -C12; Rha-Rha-C12-C12; Rha-Rha-C12-C14; Rha-Rha-C14-012; Rha-Rha- 014-014; Rha-Rha-C14-C16; Rha-Rha-C16-C14; Rha-Rha-C16-016; Rha-Rha-014- 014-014; Rha-Rha-C10-C10-CH3; Decenoyl-Rha-Rha-C10-010; or combinations thereof.

[0049] The terms “active”, “% active”, and “% active weight” refer to the amount of the active ingredient without regard to the amount of water or other solvent that may be present with the ingredient.

[0050] As used herein, “effective amount” refers to an amount of an active ingredient or composition that, when administered to a wound, is capable of accelerating or otherwise facilitating the healing process, including the inhibition of biofilm formation during wound closure. The actual amount may vary depending on a number of factors,including, but not limited to, the severity of the wound, the age and health status of the subject, and the form of administration.

[0051] The mono-rhamnolipid may comprise one or more mono-rhamnolipid-mono- lipidic congeners, including for example: Rha-C8:2; Rha-C8; Rha-C10; Rha-C12:2; Rha- C12; Rha-C14:2; or combinations thereof. The mono-rhamnolipid may also comprise one or more mono-rhamnolipid-di-lipidic congeners, including for example: Rha-C8-C8; Rha- 08-010:1 ; Rha-C10:1 -C8; Rha-C8-C10; Rha-C10-C8; Rha-C10-C10:1 ; Rha-C10-C10; Rha-C8-C12; Rha-C12-C8; Rha-C10-C12:1 ; Rha-C12:1 -C10; Rha-C10-12; Rha-C12- C10; Rha-C10-C14:1 ; Rha-C12-C12:1 ; Rha-C10-C14; Rha-C12-C12; Rha-C12-C14; Rha-C14-C14; Rha-C14-C16; Rha-C16-C16; Rha-C10-C10-CH3; Decenoyl-Rha-C10- C10; or combinations thereof.

[0052] The di-rhamnolipid may comprise one or more di-rhamnolipid-mono-lipidic congeners, including for example: Rha-Rha-C8; Rha-Rha-C10; Rha-Rha-C12:1 ; Rha- Rha-C12; Rha-Rha-C14; or combinations thereof. The di-rhamnolipid may also comprise one or more di-rhamnolipid-di-lipidic congeners, including for example: Rha-Rha-C8-C8; Rha-Rha-C8-C10; Rha-Rha-C10-C8; Rha-Rha-C10-C10:1 ; Rha-Rha-C10-010; Rha- Rha-C8-C12:1 ; Rha-Rha-C12:1 -C8; Rha-Rha-C10-012:1 ; Rha-Rha-C12:1 -C10; Rha- Rha-C10-C12; Rha-Rha-C12-010; Rha-Rha-C10-C14:1 ; Rha-Rha-C12-C12:1 ; Rha- Rha-C12:1 -C12; Rha-Rha-C12-C12; Rha-Rha-C12-C14; Rha-Rha-C14-012; Rha-Rha- 014-014; Rha-Rha-C14-C16; Rha-Rha-C16-014; Rha-Rha-C16-016; Rha-Rha-014- 014-014; Rha-Rha-C10-C10-CH3; Decenoyl-Rha-Rha-C10-010; or combinations thereof.

[0053] In one aspect, the present technology provides a wound care composition, and methods of use, wherein the composition comprises a mixture of rhamnolipids in an amount of 0.1 % to 99% by weight, based on the total weight of the composition, wherein the mixture of rhamnolipids comprises mono-rhamnolipids and di-rhamnolipids in a weight ratio 40:60 to 60:40 mono-rhamnolipids:di-rhamnolipids, alternatively 40:60 to 50:50 mono-rhamnolipids:di-rhamnolipids, alternatively about 40:60 to about 48:52, alternatively 42:58 to 48:52 mono-rhamnolipids:di-rhamnolipids.

[0054] A further aspect of the present technology provides a method for treating a wound comprising administering to the wound an effective amount of a composition comprising a mixture of rhamnolipids, thereby accelerating or otherwise facilitating healing, wherein the mixture of rhamnolipids comprises mono-rhamnolipids and di- rhamnolipids in a weight ratio of 40:60 to 60:40 mono-rhamnolipids:di-rhamnolipids, alternatively 40:60 to 50:50 mono-rhamnolipids:di-rhamnolipids, alternatively 40:60 to 48:52 mono-rhamnolipids:di-rhamnolipids, alternatively 42:58 to 48:52 mono- rhamnolipids:di-rhamnolipids.

[0055] In some embodiments, the present technology provides a method for treating a wound, as described above, in which the mixture of rhamnolipids comprises mono- rhamnolipids and di-rhamnolipids in a weight ratio of 42:58 to 48:52, an amount of Rha- C10-C10 mono-rhamnolipid of about 29% to about 40% by weight, and an amount of RhaRha-C10-C10 di-rhamnolipid of about 35% to 50% by weight, based on the total weight of the rhamnolipids in the mixture of rhamnolipids.

[0056] The rhamnolipids may be produced from a rhamnolipid-producing microorganism that has the capacity to synthesize / produce rhamnolipids under suitableconditions. Such microorganisms include, but are not limited to, bacteria, particularly bacteria of the phyla Pseudomonadota, Actinobacteria, Fimicutes, and Proteobacteria. The rhamnolipids are naturally derived and therefore have a BCI of 100. In a particular embodiment, the rhamnolipid-producing microorganism for producing the rhamnolipids is Pseudomonas aeruginosa. Methods of culturing the rhamnolipid-producing bacteria and the production of rhamnolipids from fermentation are known in the art from, for example U.S. Patent No 1 1 ,142,782 and U.S. Patent No. 10,144,943, incorporated herein by reference in their entirety. Methods of purifying the rhamnolipids are also known in the art from, for example, U.S. Patent No 9,884,883 and U.S. Patent No. 10,829,507, incorporated herein by reference in their entirety.

[0057] The mixture of rhamnolipid salts can be used alone, as the sole active ingredient in the wound care I treatment composition. When used alone, the mixture of rhamnolipids may be in the range of about 0.01 % to about 99% by active weight, based on the total weight of the composition, alternatively about 0.02% to about 25%, alternatively about 0.1 % to about 10%, alternatively about 0.2% to about 6% by active weight, based on the total weight of the composition. The mixture of rhamnolipid salts may be used as a coactive in combination with another active ingredient, such humectants and other skin wellness ingredients (e.g., that help promote skin wellness and improve the skin’s natural barrier properties). The mixture of rhamnolipid salts may also be used as a co-active in combination with another active ingredient, such as antibiotics, vitamins (e.g., vitamins E, A, and C), and Hyaluronic Acid. When used in combination, the mixture of rhamnolipids may be in the range of about 0.01 % to about 99% by active weight, based on the total weight of the composition, alternatively about 0.02% to about 25%, alternatively about0.1 % to about 10%, alternatively about 0.2% to about 6% by active weight, by active weight based on the total weight of the composition. The combination of the mixture of rhamnolipid salts and another co-active ingredient may help to alleviate the irritation potential of the co-active without reducing or inhibiting its activity. Combining the mixture of rhamnolipid salts with another co-active ingredient may also allow for the reduction of the other co-active ingredient, which can also help to reduce the overall irritation potential.

[0058] The wound care I treatment compositions can be formulated into any treatment form commonly used for dermatological / topical applications. For example, the compositions can be in the form of an aqueous solution, suspension, cream, lotion, gel, paste, spray, cream, foam or emollient, or impregnated onto pads, wipes, bandages and / or dressings.

[0059] The wound care / treatment compositions of the present technology also include at least one carrier suitable for wound care I treatment to bring the total percentage of the composition to 100%. As will be appreciated by at least those skilled in the art, a variety of carriers, vehicles, diluents, and the like are suitable for use in the practice of the present technology. Thus, it will also be appreciated that the terms “carrier”, “vehicle”, and “diluent” are to be considered non-exhaustive and interchangeable with respect to the present technology and in describing the various formulations, applications, uses, and compositions thereof.

[0060] Water is a suitable carrier, and can be de-ionized water, hard water, soft water, distilled water, tap water or combinations thereof. Water can be used alone as the carrier, or in combination with other carriers suitable for personal care, such as for example, alcohols such as ethanol, isopropanol, or benzyl alcohol; glycols such as propyleneglycol, or polyethylene glycol. Other carriers can include, but are not limited to solvents, emulsifiers, or solubilizers.

[0061] When the treatment form is a cream, gel, or paste, the wound care I treatment compositions can include, but are not limited to, vegetable gums, starches, celluloses, waxes, silicone, silica, or clays, as carrier ingredients. When the treatment form is a spray, the composition may include a propellant.

[0062] In addition to the rhamnolipid active and carrier, the wound care / treatment compositions of the present technology can include optional ingredients as known in the art. Such other components or additives can include, but are not limited to, surfactants, pH adjustment agents, skin conditioners, antioxidants, preservatives, fragrances, pigments, dyes, and other excipients (e.g., anesthetics such as Benzocaine, and other antibiotics).

[0063] The wound care I treatment compositions of the present technology can have pH values in the range of about 4.0 to about 8.5, alternatively, about 5.0 to about 8.0, ideally 5.5 to 7.0.

[0064] The wound care / treatment compositions of the present technology may be used by applying the composition to the wound of a subject in an amount effective to treat, and / or otherwise facilitate wound healing. “Applying” can refer to any commonly used method of application, such as, but not limited to, spreading a cream or gel containing the wound care I treatment composition on the surface of the wound and allowing the cream or gel to remain on the wound; spraying a liquid containing the wound care / treatment composition on the surface of the wound and, if desired, surrounding tissue; wiping the wound with a wipe impregnated with the wound care / treatmentcomposition and allowing the composition to remain on the wound and, if desired, surrounding tissue; applying a pad impregnated with the wound care / treatment composition and allowing the pad to remain on the surface of the wound and, if desired, surrounding tissue; or an aqueous or non-aqueous liquid wash intended to treat the surface of the wound and, if desired, the surrounding tissue.

[0065] Dosage forms and treatment regimens using the wound care / treatment compositions of the present technology can vary with the type and intensity of the wound. In one or more embodiments, methods of treatment in accordance with the present technology may use a one, two, three, four, or more daily dosage regime. The daily dosage regimen can continue for 1 -6 days, alternatively one, two, three, four, five, six, or more weeks according to the condition and response of the patient.

[0066] Methods of the present disclosure comprise applying a rhamnolipid containing wound care / treatment composition to a wound, wherein application of the composition inhibits or otherwise reduces the formation of pathogenic biofilms during wound closure.ITEMS OF THE INVENTIONThe invention further relates to the following items:1 A rhamnolipid composition for use in methods of inhibiting or reducing biofilm formation during wound closure, wherein the composition comprises a mixture of mono-rhamnolipids and di-rhamnolipids having a mono-rhamnolipids:di- rhamnolipids weight ratio of about 40:60 to about 60:40; and wherein application of the composition to a wound accelerates wound closure. The rhamnolipid composition also comprises, based on the total weight of rhamnolipids present inthe composition, an amount of 010-010 mono-rhamnolipid of about 29% to about 40% by weight, an amount of 010-010 di-rhamnolipid of about 35% to about 50% by weight, an amount of C8-C10 mono-rhamnolipid of about 2% to about 5% by weight; an amount of 08-010 di-rhamnolipid of about 2% to about 5% by weight; an amount of 010-012 mono-rhamnolipid of about 2% to about 6% by weight; and an amount of 010-012 di-rhamnolipid of about 8% to about 14% by weight. The composition also further comprises at least one acceptable carrier, and optionally one or more additives, in an amount to total 100% by weight of the composition.2. The composition of the preceding item, wherein the mixture of rhamnolipids comprises an amount of total mono rhamnolipid of about 40% to about 50% by weight, preferably about 40% to about 48% by weight, such as about 42 % to about 48% by weight, or about 43% to about 47% by weight, or about 43% to about 45% by weight, based on the total weight of rhamnolipids present in the composition.3 The composition of any one of the preceding items, wherein the mixture of rhamnolipids comprises an amount of total di-rhamnolipid of about 50% to about 60% by weight, preferably about 52% to about 60% by weight, such as about 52% to about 58% by weight, or about 53% to about 57% by weight, or about 55% to about 57% by weight, based on the total weight of rhamnolipids present in the composition.4 The composition of any one of the preceding items, wherein the mixture of rhamnolipids comprises an amount of the mono-rhamnolipid C8-C10 congener in the range of about 2% to about 4% by weight, based on the total weight of rhamnolipids present in the composition.5 The composition of any one of the preceding items, wherein the mixture of rhamnolipids comprises an amount of the mono-rhamnolipid C10-C10 congener in the range of about 29% to about 37% by weight, preferably about 32% to about 37% by weight, such as about 34% to about 37% by weight, or about 35% to about 37% by weight, based on the total weight of rhamnolipids present in the composition.6 The composition of any one of the preceding items, wherein the mixture of rhamnolipids comprises an amount of the mono-rhamnolipid C10-C12 congener in the range of about 2% to about 5% by weight, preferably about 3.5% to about 5% by weight, based on the total weight of rhamnolipids present in the composition.7 The composition of any one of the preceding items, wherein the mixture of rhamnolipids comprises an amount of the di-rhamnolipid C8-C10 congener in the range of about 2% to about 4% by weight, based on the total weight of rhamnolipids present in the composition.8 The composition of any one of the preceding items, wherein the mixture of rhamnolipids comprises an amount of the di-rhamnolipid C10-C10 congener in the range of about 35% to about 45% by weight, preferably about 36% to about 40% by weight, such as about 36% to about 38% by weight, based on the total weight of rhamnolipids present in the composition.9 The composition of any one of the preceding items, wherein the mixture of rhamnolipids comprises an amount of the di-rhamnolipid 010-C12.1 congener in the range of about 2% to about 5% by weight, preferably about 3% to about 5% byweight, such as about 3% to about 4% by weight, based on the total weight of rhamnolipids present in the composition.10 The composition of any one of the preceding items, wherein the mixture of rhamnolipids comprises an amount of the di-rhamnolipid C10-C12 congener in the range of about 9% to about 12% by weight, preferably in the range of about 10% to about 12% by weight, or about 10% to about 12.5% by weight, based on the total weight of rhamnolipids present in the composition.1 1 . The composition of any one of the preceding items, wherein the mixture of rhamnolipids comprises an amount of the di-rhamnolipid C12-C12 congener in the range of about 0.1 % to about 0.5% by weight, preferably about 0.2% to about 0.4% by weight, such as about 0.2% to about 0.3% by weight, or about 0.25% to about 0.3% by weight, based on the total weight of rhamnolipids present in the composition.12. The composition of any one of the preceding items, wherein the mixture of rhamnolipids is a SEP-RM Rhamnolipid composition.13. The composition of any one of the preceding items, wherein the mixture of rhamnolipids is a SEP-RM Rhamnolipid composition obtained by a solvent extraction process including a bleaching step after acidulation and before the solvent extraction.EXAMPLES

[0067] The presently described technology and its advantages will be better understood by reference to the following examples. These examples are provided todescribe specific embodiments of the present technology. By providing these examples, the inventors do not limit the scope and spirit of the present technology.Experimental Procedures***Test MaterialsRhamnolipid Compositions

[0068] Rhamnolipids are produced, as understood in the art, through the fermentation of rhamnolipid producing bacteria such as Pseudomonas aeruginosa. The rhamnolipids may be produced from a rhamnolipid-producing microorganism that has the capacity to synthesize / produce rhamnolipids under suitable conditions. Such microorganisms include, but are not limited to, bacteria, particularly bacteria of the phyla Pseudomonadota, Actinobacteria, Fimicutes, and Proteobacteria. The rhamnolipids are naturally derived and therefore have a BCI of 100. In a particular embodiment, the rhamnolipid-producing microorganism for producing the rhamnolipids is Pseudomonas aeruginosa. Methods of culturing the rhamnolipid-producing bacteria and the production of rhamnolipids from fermentation are known in the art from, for example U.S. Patent No 1 1 ,142,782 and U.S. Patent No. 10,144,943, incorporated herein by reference in their entirety. Methods of purifying the rhamnolipids are also known in the art from, for example, U.S. Patent No 9,884,883 and U.S. Patent No. 10,829,507, incorporated herein by reference in their entirety.

[0069] Fermentation whole broth then undergoes multiple purification steps. The specific combination of steps depends on the rhamnolipid actives and purity requirements of the target application. Certain rhamnolipid mixtures used herein are purified by a solvent extraction process. As used herein, the acronym SEP-RM refers to SolventExtraction Purified Rhamnolipid Mixture (or composition), in accordance with the current specification.

[0070] For example, the SEP-RM Rhamnolipid compositions used herein are prepared in accordance with at least the following processing steps: (1 ) fermentation of appropriate Rhamnolipid producing bacterium; (2) biomass separation; (3) sterilization; (4) clarification (e.g. filtration); (5) acidulation; (6) bleaching; (7) washing; (8) solvent extraction; (9) neutralization and dilution; and (10) final polishing step(s). As discussed below, the solvent extraction process / step is intended to yield a higher purity rhamnolipid mixture / composition for personal care applications. The additional solvent extraction steps provide a rhamnolipid composition having a lighter color and milder odor profile that are preferred for the personal care market.

[0071] At the end of fermentation, the whole broth typically contains rhamnolipids along with biomass and other by-products of fermentation. To separate the biomass solids, the broth can be centrifuged. The resulting centrifuged broth is then subjected to sterilization (e.g., high temperature sterilization), after which, the centrifuged sterilized broth is clarified by filtration to remove suspended solids.

[0072] Further purification is achieved by treating the clarified broth with acid, which converts the rhamnolipid to a water-insoluble form that settles to the bottom and separates from the bulk aqueous phase. This dense acidulated rhamnolipid is then isolated (and referred to as Acidulated, Concentrated Clarified Broth (ACCB). ACCB is then treated with bleaching agent, then water washed to remove residual bleaching agent and other water-soluble impurities. At this point, the decolorized washed ACCB isconcentrated (e.g., > about 45% actives, or between about 35% to about 55% actives) and purer (e.g., > about 75% purity, or between about 65% to about 85% purity).

[0073] Solvent Extraction Process - To achieve better purity, color, and odor, the decolorized Acidulated, Concentrated Clarified Broth (ACCB) undergoes a solvent extraction process. This is generally performed by dissolving the decolorized, washed ACCB in organic solvent, preferably ethyl acetate. The rag layer that typically forms is separated from the bulk solution. Activated carbon is then added to the rhamnolipid solution in ethyl acetate for further decolorization and deodorization. The slurry is filtered and the resulting solution is stripped under vacuum to remove ethyl acetate. The highly concentrated ACCB obtained as residue is then neutralized and diluted (e.g., from between about 20% to about 30% actives, preferably about 25% actives) to give crude SEP-RM. As a final polishing step, crude SEP-RM is then washed with ethyl acetate to extract the antifoam and yield the final product as optically clear SEP-RM at about 25% active and about 85% purity.Mono-Rhamnolipid:Di-Rhamnolipid Ratio and Congener Distribution

[0074] The mono-rhamnolipid:di-rhamnolipid ratio, and congener distribution for SEP- RM rhamnolipid compositions of the present disclosure can be determined using HPLC (High Performance Liquid Chromatography). As understood by the person of ordinary skill in the art, HPLC will separate rhamnolipid congeners by chain length and number of rhamnose moieties (with the more polar congeners eluting first). Fractions are collected and analyzed by mass spectrometry to identify each peak. The congener distribution is reported as area percent (with actives being calculated relative to known standards).

[0075] The HPLC congener distribution for SEP-RM rhamnolipid compositions of the present technology may comprise mono-rhamnolipid C8-C10 congener in an amount of about 2% to about 5% by weight, di-rhamnolipid C8-C10 congener in an amount of about 2% to about 5% by weight, mono-rhamnolipid C10-C10 congener in an amount of about 29% to about 40% by weight, preferably about 35% to about 40% by weight, di- rhamnolipid C10-C10 congener in an amount of about 35% to about 50 weight, preferably about 35% to about 45% by weight, mono-rhamnolipid C10-C12 congener in an amount of about 2% to about 6% by weight, di-rhamnolipid 010-012 congener in an amount of about 8% to about 14% by weight, with total mono-rhamnolipid congeners in an amount of about 40% to about 48% by weight, and total di-rhamnolipid congeners in an amount of about 52% to about 60% by weight, based on the total weight of the congeners in the composition.

[0076] The HPLC congener distribution for an exemplary SEP-RM rhamnolipid composition of the present disclosure, is presented below:Optional Bleaching Step

[0077] The SEP-RM rhamnolipid composition provided herein can be processed either with, or without a bleaching step (occurring after acidulation, and before the solvent extraction step(s)). For the SEP-RM rhamnolipid compositions of the present disclosure, the mono-rhamnolipid:di-rhamnolipid ratio, and the associated congener distribution, are unaffected by whether a bleaching step is included, or not included, in the purification process.Example 1

[0078] This study determined how certain rhamnolipid compositions impacted the development and / or inhibition of biofilm during wound healing / closure.

[0079] The objective of the study was to evaluate various concentrations (e.g., 1 % and 3%) of a SEP-RM Rhamnolipid gel in an advanced wound care prototype. The animal model used was the male BKS.Cg-Dock7m + / + Lepr db / J {J ax Stock # 000642; Genotype: Homozygous genotype, Wildtype for Dock7<m>, Homozygous for Lepr<db>) diabetic mouse delayed wound healing model. The study design, using SEP-RM Rhamnolipid, is summarized below in Table 1 .Table 1 : Diabetic Mouse Wound Model Study Designmg = milligram; mL = milliliter*n=3 (sacrificed during inflammatory phase, Day 3-5) n=3 sacrificed during proliferative phase, Day 1 1 -18

[0080] As indicated in Table 1 , wound creation, on Day 0, included a skin biopsy (10 mm disc) removed from the dorsal right side of the mouse model. The study included 6 treatment groups, including: 2 x control groups (without rhamnolipid); 2 x 1 % SEP-RM rhamnolipid (dose level = 0.1 mg; dose concentration = 10 mg / mL, 1 %; and dose volume = 100 uL); and 2 x 3% SEP-RM rhamnolipid (dose level = 0.2 mg; dose concentration = 20 mg / mL, 20%; and dose volume = 100 uL). The douse route was topical (to the area of the skin biopsy), at days 0, 3, 7, 10, 14, 17, and 21 . The study end was set at 24 days.

[0081] The Control group included Control Gel Collagen, with ambient storage. Rhamnolipid treatment group 1 comprised 1 % SEP-RM rhamnolipid in collagen hydrogel, with ambient storage. Rhamnolipid treatment group 2 comprised 3% SEP-RM rhamnolipid in collagen hydrogel, with ambient storage. The wound dressing comprised 3M Tegaderm™ dressing, with ambient storage.

[0082] As indicated above, the test system included species Mus muscuus (strain = BKS.Cg-Dock7m + / + Leprdb / J). The source was Jackson Laboratories (Stock# 000642; Genotype: Homozygous genotype, Wildtype for Dock7<m>, Homozygous for Lepr<db>). The age of the animals upon arrival was 8 weeks, and before use the animals were held for a minimum of 3 days for environmental acclimation.

[0083] With respect to the environment, animal holding rooms’ environmental controls were set to maintain temperatures between 20 - 26QC (68 - 79QF) with a relative humidity range of 30% - 70%. A 12-hour light / 12-hour dark cycle was maintained, except when interrupted to accommodate study procedures. Ten or greater air changes per hour were also maintained in the animal holding and procedure rooms.

[0084] With respect to diet, the test animals were provided Teklad Global 18% Protein Rodent Diet 2918 ad libitum daily. The test animals were also provided filtered tap water during the acclimation period and Innovive bottled water throughout the study period, ad libitum.

[0085] Light anesthesia inhalant isoflurane on O2 (1 - 4%, to effect) was used to facilitate wound creation, and for treatments, wound assessments, wound measurements, photography, and reapplication of dressings. Isoflurane overdose or carbon dioxide asphyxiation was used, to effect, for terminal tissue collections.

[0086] All animals had a wound created on Day 0 while anesthetized, and after received analgesics. With respect to pre-wound care, all test animals had their dorsum shaved prior to study start. All animals recieved a subcutaneous injection of Buprenorphine-SR analgesia (1.0 mg / kg) on Day 0, ~ 1 hour prior to wounding, and an additional dose 48-72 hours later, if needed.

[0087] With respect to wound creation, sterile techniques were used. The test animals were lightly anesthetized using isoflurane on O2 (1 - 4%, to effect) and placed on their abdomen. Using sterile, disposable skin biopsy punches, a 10 mm disc skin biopsy was removed from the dorsal right side of each test animal. A 3D Printed biofilm (S. aureus) was placed in the wounds of animals in groups 2A and 3A only. The wound was covered with Tegaderm™ and the biofilm was allowed to incorporate into the wound until treatment initiation (Day 3). From Day 0 until study conclusion all wounds were covered with a Tegaderm™ dressing as outlined above. Tegaderm™ was left on for the duration of the study.

[0088] Bioburden was assessed during the study period utilizing a MolecuLight i:x™ device at each dressing change, and at study end by Colony Forming Units (CFU) quantification of harvested wounds.

[0089] With respect to the dose regimen, topical administration of SEP-RM rhamnolipid treatment was performed twice per week, on Study Days 0 (non-infected groups only), 3, 7, 10, 14, 17, and 21 while animals were under light anesthesia inhalant isoflurane on O2 (1-4%, to effect). Treatment administration for the infected groups began on Study Day 3 and continued twice per week outlined above. The test animals were observed for anyadverse reactions to each dose, at each session, prior to being placed back into their respective home cages.

[0090] At the time of wound measurements and assessment, on Days 0, 3, 7, 10, 14, 17, and 21 , animals, while lightly anesthetized via isoflurane on O2 (1 - 4%, to effect), had wounds imaged with the MolecuLight i:x™ device which detected wounds with elevated bacterial loads of >104CFU / g at up to 0.8mm below the skin surface. Results from this analysis are illustrated in Figure 1 .Example 2

[0091] A study was conducted to determine the ability of a polyquaternium-1 and rhamnolipid to prevent and / or dissociate a Pseudomonas aeruginosa (ATCC#15442) and MRSA pvl gene positive - Methicillin resistant Staphylococcus aureus (ATCC# BAA 1680).Prevention method:

[0092] Prevention method: biofilm prevention refers to inhibiting the early stages of biofilm development by preventing microbial cells from attaching to a surface, such as a Calgary Biofilm Device.

[0093] For the prevention method, all samples were diluted in a nutrient medium and inoculated with a minimum of 1 .0E+07 CFU / mL.

[0094] For the MRSA (ATCC BAA1680) biofilm study, controls were Tryptic Soy Broth (TSB), Phosphate Buffer Dilution Blank (PBDB), Sterile Deionized water. Test samples included the benchmark, Plurogel (water soluble gel for burns and wounds 0.5x diluted inTryptic soy broth), 0.5% Rhamnolipid (provided at 1.0% and diluted 1 :2 in Tryptic soy broth) and 0.5% Polyquaternium-1 (provided at 1 .0% and diluted 1 :2 in T ryptic soy broth).

[0095] For the Pseudomonas aeruginosa (ATCO 15442) biofilm study, controls were Tryptic Soy Broth (TSB), Phosphate Buffer Dilution Blank (PBDB), Sterile Deionized water. Test samples included the benchmark, Betasept (4% Chlorhexadine gluconate used in wound cleaning and skin cleansing, surgical scrub - was diluted 1 :2 in Tryptic soy broth), 0.5% Rhamnolipid (provided at 1 .0% and diluted 1 :2 in Tryptic soy broth) and 0.5% Polyquaternium-1 (provided at 1.0% and diluted 1 :2 in Tryptic soy broth).

[0096] The samples were incubated in a Cellulose-Coated Calgary Biofilm Device (Innovotech: 19171 ) at 33.0 ± 2.0°C on a shaker platform for a minimum of 24 hours to allow biofilm growth to occur. Aliquots were removed and neutralized by dilution in a buffer containing flow cytometry dyes , SYTO 9 and propidium iodide. All cells were characterized by morphology and viability relative to positive and negative controls by using flow cytometry. A stain using crystal violet was conducted to qualitatively detect presence and absence of the biofilm.

[0097] All controls and test samples stated above were diluted 1 :2 (1 mL control or test samples + 1 mL sterile Tryptic soy broth). The nutrient medium is Tryptic Soy Broth (TSB). This is a well-recognized growth medium for microbial cultivation.

[0098] Inside of the Calgary Biofilm Device, the entire outer ring of all well plates was given sterile tryptic soy broth only. (Figure 2.) The inner wells (in 10 replicates per control or sample) were diluted in the nutrient as stated above and transferred aseptically to the biofilm device (first row is the nutrient, second row is PBDB, third row is water, fourth - sixth row are test samples). The microbial challenge organism at the appropriate titer wasadded to the control and sample rows of the Calgary Biofilm Device and mixed via micropipette. The lid (having pegs) was placed on the plate to limit open exposure.

[0099] Inoculated includes introducing the challenge organism to the controls and test samples. This case refers specifically to the introduction of the microbe to the rows containing the control and test samples in the Calgary Biofilm Device. The inoculation volume remained the same for all tests (1 part inoculum to 9 parts control or sample; with a minimum of 1.0E+07 CFU / mL).

[0100] The samples were incubated in a Cellulose-Coated Calgary Biofilm Device (Innovotech: 19171 ) at 33.0 ± 2.0°C on a shaker platform for a minimum of 24 hours to allow biofilm growth to occur.

[0101] Aliquots (were removed and neutralized by dilution in a buffer containing flow cytometry dyes, SYTO 9 and propidium iodide. The size of the aliquot was 30pL, which was removed via micropipette and transferred to 270pL of flow buffer (i.e. , 1 part aliquot to 9 parts flow buffer + dyes). The buffer is a “Flow Buffer,” and is a sterile media combination of Phosphate Buffer Stock (PBS), Tween 20, and Ethylenediaminetetraacetic acid (EDTA). The dyes are at a concentration of 0.84 pM SYTO9 and 66.67pM Propidium Iodide.

[0102] All cells were characterized by morphology and viability relative to positive and negative controls by using flow cytometry. Typically there is one morphology per organism (i.e., the Pseudomonas and Staphylococcus will vary between each other). The morphology provides information about the size of the cells (i.e., the flow cytometer can estimate cell size from how it scatters the light lasers), which ensures there is anuncontaminated challenge organism and the recovery of a healthy microbe (i.e., it is actually present).

[0103] A stain using crystal violet was conducted to qualitatively detect presence and absence of the biofilm. Crystal Violet is a purple dye that is widely-utilized for biofilm staining. The dye which is positively charged binds very strongly to the negative components of the extracellular polymeric substances (EPS) in the biofilm. The crystal violet stain binds to the components of the biofilm and enables qualitative visualization of the biofilm on the pegs of the Calgary Biofilm Device, with the cells / biofilm remaining on the bottom of each corresponding well in the well plate. (Figure 3.)Dissociation method:

[0104] The dissociation method focuses on the disruption of an established biofilm. Dissociation refers to disruption of a mature biofilm, wherein cells detach and revert to a planktonic state, either individually or in clusters. These planktonic cells are more susceptible to antimicrobial agents.

[0105] As with the Prevention method discussed above, for the Dissociation method a nutrient medium was inoculated (with an inoculated with a minimum of 1 .0E+07 CFU / mL) in a Cellulose-Coated Calgary Biofilm Device (Innovotech: 19171 ), and incubated at 33.0 ± 2.0°C on a shaker platform for 24 hours to allow biofilm growth to occur. All remaining medium was aspirated and test samples were diluted in a nutrient medium and incubated at 33.0 ± 2.0°C on a shaker platform for 24 hours to allow biofilm growth to occur. Aliquots were removed and neutralized by dilution in a buffer containing flow cytometry dyes, SYTO 9 and propidium iodide. All cells were characterized by morphology and viabilityrelative to positive and negative controls using flow cytometry. A stain using crystal violet was conducted to qualitatively detect presence and absence of the biofilm.Time Kill Study

[0106] Using the Prevention and Dissociation methods set forth above, a study was conducted to evaluate the time-dependent antimicrobial efficacy of polyquaternium-1 and rhamnolipid against Pseudomonas aeruginosa (ATCC# 15442). Test samples were prepared by diluting the test samples in sterile water from concentrations of 1 .25% - 5.0%. A combination of the rhamnolipid and polyquaternium-1 were evaluated at concentrations of rhamnolipid / polyquaternium-1 ratios of 1 :1 and 3:1 (1 .25%:1 .25%, 2.5%:2.5%, 3.75%:1 .25%) to evaluate a further synergistic relationship of efficacy. Test samples were inoculated with a minimum of 1 .0E+08 CFU / mL of the test system and incubated at 33.0 ± 2.0°C (to mimic skin temperature) for a minimum of 2 hours. Aliquots were removed and neutralized by dilution in a buffer containing flow cytometry dyes SYTO 9 and propidium iodide. All cells were characterized by morphology and viability relative to positive and negative controls using flow cytometry.

[0107] Figure 4 shows the Log Reduction of MRSA viable cells using the Prevention and Dissociation methods set forth above.

[0108] Figure 5 graphically shows the Log Reduction of Methicillin resistantStaphylococcus aureus viable cells using the Prevention method set forth above.

[0109] Figure 6 graphically shows the Log Reduction of Methicillin resistantStaphylococcus aureus viable cells using the Dissociation method set forth above.

[0110] Figure 7 shows the Log Reduction of P. aeruginosa viable cells using thePrevention and Dissociation methods set forth above.

[0111] Figure 8 graphically shows the Log Reduction of P. aeruginosa viable cells after 24 hours using the Dissociation method set forth above.

[0112] Figure 9 graphically shows the Log Reduction of P. aeruginosa viable cells after 24 hours using the Prevention method set forth above.

[0113] The present disclosure provides for the prevention and / or dissociation (disruption) of biofilm with the application of rhamnolipid compositions, in accordance with the present disclosure. Also, the present disclosure provides for the prevention and / or dissociation (disruption) of biofilm with the application of Polyquaternium-1 (PQ-1 ) compositions, in accordance with the present disclosure.

[0114] In one aspect, the application of rhamnolipids or Polyquaternium-1 , in accordance with the present disclosure, provides for the dissociation / disruption of biofilm. In this aspect, the rhamnolipid is present at a concentration of 0.5 wt%, from 0.5 - 2.0 w%, from 0.10 - 5.0 wt%, or from 0.01 - 10.0 wt%, based on the weight of the total composition. Also, in this aspect, the Polyquaternium-1 is present at a concentration of 0.5 wt%, from 0.5 - 2.0 w%, from 0.10 - 5.0 wt%, or from 0.01 - 10.0 wt%, based on the weight of the total composition.

[0115] In another aspect, the application of rhamnolipids or Polyquaternium-1 , in accordance with the present disclosure, provides for the prevention of biofilm. In this aspect, the rhamnolipid is present at a concentration of 0.5 wt%, from 0.5 - 2.0 w%, from 0.10 - 5.0 wt%, or from 0.01 - 10.0 wt%, based on the weight of the total composition.Also, in this aspect, the Polyquaternium-1 is present at a concentration of 0.5 wt%, from 0.5 - 2.0 w%, from 0.10 - 5.0 wt%, or from 0.01 - 10.0 wt%, based on the weight of the total composition.

[0116] In another aspect, the application of rhamnolipids in accordance with the present disclosure provides a Log Reduction (of microbial biofilm) of 1.75, from 1.5 to 1 .75, from 1 .25 to 1 .75, from 1 .0 to 1 .75, from 1 .75 to 2.0, from 1 .75 to 2.25, or from 1 .75 to 2.5. In this aspect, the recited Log Reduction is within 24 hours, 36 hours, or 48 hours.

[0117] In another aspect, the application of rhamnolipids in accordance with the present disclosure provides for the prevention of biofilm formation, including a Log Reduction (of microbial biofilm) of 1.75, from 1.5 to 1.75, from 1.25 to 1.75, from 1.0 to 1 .75, from 1 .75 to 2.0, from 1 .75 to 2.25, or from 1 .75 to 2.5 (see, e.g., Prevention Method set forth above). In this aspect, the recited Log Reduction is within 24 hours, 36 hours, or 48 hours.

[0118] In a still further aspect, the application of rhamnolipids in accordance with the present disclosure provides for the dissociation / disruption of a formed biofilm, including a Log Reduction (of microbial biofilm) of 1.75, from 1.5 to 1.75, from 1.25 to 1.75, from 1 .0 to 1 .75, from 1 .75 to 2.0, from 1 .75 to 2.25, or from 1 .75 to 2.5 (see, e.g., Dissociation Method set forth above). In this aspect, the recited Log Reduction is within 24 hours, 36 hours, or 48 hours.

[0119] In another aspect, the application of Polyquaternium-1 (PQ-1 ) in accordance with the present disclosure provides for the prevention of biofilm formation, including a Log Reduction (of microbial biofilm) of 1.5, from 1.5 to 1.75, from 1.25 to 1.75, from 1.0 to 1 .75, from 1 .75 to 2.0, from 1 .5 to 2.0, from 1 .5 to 2.25, or from 1 .5 to 2.5 (see, e.g.,Prevention Method set forth above). In this aspect, the recited Log Reduction is within 24 hours, 36 hours, or 48 hours.

[0120] In a still further aspect, the application of Polyquaternium-1 (PQ-1 ) in accordance with the present disclosure provides for the dissociation / disruption of a formed biofilm, including a Log Reduction (of microbial biofilm) of 2.25, from 1 to 2.25, from 1.25 to 2.25, from 1.5 to 2.25, from 1.0 to 2.25, or from 2.0 to 2.5, (see, e.g., Dissociation Method set forth above). In this aspect, the recited Log Reduction is within 24 hours, 36 hours, or 48 hours.

[0121] The present technology is now described in such full, clear and concise terms as to enable a person skilled in the art to which it pertains, to practice the same. It is to be understood that the foregoing describes preferred embodiments of the present technology and that modifications may be made therein without departing from the spirit or scope of the present technology as set forth in the appended claims. Further, the examples are provided to not be exhaustive but illustrative of several embodiments that fall within the scope of the claims.

Claims

Claims1 . A composition for use in a method of inhibiting biofilm formation during wound closure, the composition comprising:(a) a mixture of rhamnolipids, wherein the mixture of rhamnolipids comprises: mono-rhamnolipids and di-rhamnolipids in a weight ratio of about 40:60 to about 60:40 mono-rhamnolipids:di-rhamnolipids; an amount of C10-C10 mono-rhamnolipid of about 29% to about 40% by weight, based on the total weight of rhamnolipids present in the composition; an amount of C10-C10 di-rhamnolipid of about 35% to about 50% by weight, based on the total weight of rhamnolipids present in the composition; an amount of C8-C10 mono-rhamnolipid of about 2% to about 5% by weight, based on the total weight of rhamnolipids present in the composition; an amount of C8-C10 di-rhamnolipid of about 2% to about 5% by weight, based on the total weight of rhamnolipids present in the composition; an amount of C10-C12 mono-rhamnolipid of about 2% to about 6% by weight, based on the total weight of rhamnolipids present in the composition; and an amount of C10-C12 di-rhamnolipid of about 8% to about 14% by weight, based on the total weight of rhamnolipids present in the composition;(b) at least one acceptable carrier, and optionally one or more additives, in an amount to total 100% by weight of the composition.

2. A method for removing biofilms from chronic and acute wounds, the method comprising the administration of a rhamnolipid containing composition comprising:(a) a mixture of rhamnolipids, wherein the mixture of rhamnolipids comprises: mono-rhamnolipids and di-rhamnolipids in a weight ratio of about 40:60 to about 60:40 mono-rhamnolipids:di-rhamnolipids; an amount of C10-C10 mono-rhamnolipid of about 29% to about 40% by weight, based on the total weight of rhamnolipids present in the composition; an amount of C10-C10 di-rhamnolipid of about 35% to about 50% by weight, based on the total weight of rhamnolipids present in the composition; an amount of C8-C10 mono-rhamnolipid of about 2% to about 5% by weight, based on the total weight of rhamnolipids present in the composition; an amount of C8-C10 di-rhamnolipid of about 2% to about 5% by weight, based on the total weight of rhamnolipids present in the composition; an amount of C10-C12 mono-rhamnolipid of about 2% to about 6% by weight, based on the total weight of rhamnolipids present in the composition; and an amount of C10-C12 di-rhamnolipid of about 8% to about 14% by weight, based on the total weight of rhamnolipids present in the composition;(b) at least one acceptable carrier, and optionally one or more additives, in an amount to total 100% by weight of the composition.

3. The composition for use in a method of inhibiting biofilm formation of claim 1 or removing biofilms claim 2, wherein the mixture of rhamnolipids further comprises an amount of 010-012:1 di-rhamnolipid of about 2% to about 5% by weight, based on the total weight of rhamnolipids present in the composition.

4. The composition for use in a method of inhibiting biofilm formation of claim 1 or removing biofilms claim 2, wherein the mixture of rhamnolipids further comprises an amount of C12-C12 di-rhamnolipid of about 0.2% to about 0.4% by weight, based on the total weight of rhamnolipids present in the composition.

5. The composition for use in a method of inhibiting biofilm formation of claim 1 or removing biofilms claim 2, wherein the mixture of rhamnolipids comprises an amount of total mono-rhamnolipid of about 40% to about 50% by weight, based on the total weight of rhamnolipids present in the composition.

6. The composition for use in a method of inhibiting biofilm formation of claim 1 or removing biofilms claim 2, wherein the mixture of rhamnolipids comprises an amount of total di-rhamnolipid of about 50% to about 60% by weight, based on the total weight of rhamnolipids present in the composition.

7. The composition for use in a method of inhibiting biofilm formation of any one of claims 1 to 5, wherein the mixture of rhamnolipids is in an amount of about 0.1 % to about 10%, based on the total weight of the composition.

8. The composition for use in a method of inhibiting biofilm formation of any one of claims 1 to 7, wherein the wound being treated is an incision, laceration, abrasion, or burn.

9. The composition for use in a method of treating a chronic wound of any one of claims 1 to 7, wherein the chronic wound being treated is diabetic foot ulcer (DFU), pressure ulcer (PU), or venous leg ulcer (VLU).

10. The composition for use in a method of inhibiting biofilm formation of any one of claims 1 to 9, wherein the at least one acceptable carrier is water alone or in combination with an alcohol or glycol.1 1 . The composition for use in a method of inhibiting biofilm formation of claim 10, wherein the alcohol is ethanol, isopropanol, or benzyl alcohol.

12. The composition for use in a method of inhibiting biofilm formation of claim 10, wherein the glycol is propylene glycol or polyethylene glycol.

13. The composition for use in a method of inhibiting biofilm formation of any one of claims 1 to 12, wherein the method comprises topical administration of the composition.

14. A method of inhibiting biofilm formation or removing biofilms to promote wound healing, comprising: applying to a wound a composition comprising:(a) a mixture of rhamnolipids, wherein the mixture of rhamnolipids comprises: mono-rhamnolipids and di-rhamnolipids in a weight ratio of about 40:60 to about 50:50 mono-rhamnolipids:di-rhamnolipids; an amount of C10-C10 mono-rhamnolipid of about 29% to about 37% by weight, based on the total weight of rhamnolipids present in the composition; an amount of C10-C10 di-rhamnolipid of about 35% to about 50% by weight, based on the total weight of rhamnolipids present in the composition; an amount of C8-C10 mono-rhamnolipid of about 2% to about 5% by weight, based on the total weight of rhamnolipids present in the composition; an amount of C8-C10 di-rhamnolipid of about 2% to about 5% by weight, based on the total weight of rhamnolipids present in the composition; an amount of C10-C12 mono-rhamnolipid of about 2% to about 6% by weight, based on the total weight of rhamnolipids present in the composition; and an amount of C10-C12 di-rhamnolipid of about 8% to about 14% by weight, based on the total weight of rhamnolipids present in the composition;(b) at least one acceptable carrier, and optionally one or more additives, in an amount to total 100% by weight of the composition wherein the composition inhibits formation of a biofilm during closure of the wound.

15. The wound care composition of claim 14, wherein the mixture of rhamnolipids further comprises an amount of C10-C12:1 di-rhamnolipid of about 2% to about 5% by weight, based on the total weight of rhamnolipids present in the composition.

16. The method of claim 14 or 15, wherein the mixture of rhamnolipids further comprises an amount of C12-C12 di-rhamnolipid of about 0.2% to about 0.4% by weight, based on the total weight of rhamnolipids present in the composition.

17. The method of any one of claims 14-15, wherein the mixture of rhamnolipids comprises an amount of total mono-rhamnolipid of about 40% to about 48% by weight, based on the total weight of rhamnolipids present in the composition.

18. The method of any one of claims 14-15, wherein the mixture of rhamnolipids comprises an amount of total di-rhamnolipid of about 52% to about 60% by weight, based on the total weight of rhamnolipids present in the composition.

19. The method composition of any one of claims 14-18, wherein the mixture of rhamnolipids is in an amount of about 0.1 % to about 10%, based on the total weight of the composition.

20. The method of any one of claims 14-19, wherein the at least one acceptable carrier is water alone or in combination with an alcohol or glycol.21 . The method of claim 20, wherein the alcohol is ethanol, isopropanol, or benzyl alcohol.

22. The method of claim 20, wherein the glycol is propylene glycol or polyethylene glycol.

23. The method of claim 2, where the rhamnolipid containing composition can reduce the level of a multidrug resistant organisms (MDRO) by <5 logs (from 109colony forming units (CFU) per wound to <104CFU) within a week of treatment.

24. The method of claim 23, where the MDRO is MRSA, VRE, or common wound pathogen (Pseudomonas aeruginosa Streptococcus pyogenes, Enterobacter cloacae, Escherichia coli, Corynebacteria, Proteus mirabilis, Proteus vulgaris, and Morganella morganii).

25. The method of claim 2, wherein the rhamnolipid containing composition disrupts the extracellular polymeric substance (EPS) to destabilize or remove the biofilm.

26. The method of claim 25, wherein the EPS component removed is either microbe, protein, lipid, polysaccharide or extracellular DNA (eDNA).

27. The method of claim 25, wherein removing the biofilm allows the chronic wound to proceed to proceed to the normal healing process.Would it be beneficial to include anywhere in here a discussion about the failure of some current SOC methods to completely eliminate biofilm which then leads to recurrent infection and things like the MMP feedback loop that overall. While necessary to the healing process, the elevated response due to infection can significantly impede the healing process and therefore its use can address these shortcomings.

Images