Rhamnosomes and rhamnolipid modified liposomes

Abstract

Disclosed are compositions comprising rhamnolipids for use in the preparation of rhamnosomes. Also disclosed are methods for preparing rhamnosomes, and for the use of these rhamnosomes in, for example, the agricultural and personal care industries.

Description

Docket No. 68255WO01RHAMNOSOMES AND RHAMNOLIPID MODIFIED LIPOSOMESCROSS-REFERENCE TO RELATED APPLICATIONSThis application claims priority to United States Provisional Application No. 63 / 684918, filed August 20, 2024, and to United States Provisional Application No. 63 / 719956, filed November 13, 2024. The entire specifications of both provisional applications referred to above are hereby incorporated by reference.BACKGROUND OF THE INVENTION

[0001] The present technology, in general, relates to methods and compositions for the formation of rhamnosomes and rhamnolipid modified liposomes. More particularly, the present technology relates to rhamnosomes and rhamnolipid modified liposomes for actives delivery in personal care.

[0002] Limitations and disadvantages of traditional liposomes will become apparent to one of skill in the art, through comparison of such liposomes with certain aspects of the rhamnosomes and rhamnolipid modified liposomes set forth in the remainder of this disclosure. The current disclosure overcomes these limitations and disadvantages, including, for example, liposomal instability, actives instability, the difficulty and expense to scale fabrication, and limited skin penetration of encapsulated actives.

[0003] What is more, there has 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. 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.BRIEF SUMMARY OF THE INVENTION

[0004] In one aspect, the present technology provides for rhamnosomes, and rhamnolipid modified liposomes, for the enhanced delivery of actives, including for example in the personal care, nutraceutical, pharmaceutical, and agricultural industries. The rhamnosomes and rhamnolipid modified liposomes disclosed herein comprise at least one rhamnolipid, including purified mono-rhamnolipids, purified di-rhamnolipids, and rhamnolipid mixtures comprising both mono- and di-rhamnolipids.

[0005] The rhamnosomes and rhamnolipid modified liposomes disclosed herein can also comprise at least one phospholipid. The phospholipid is generally selected from phosphatidylcholines, phosphatidylethanolamines, phosphatidic acid, phosphatidylserines, phosphoinositides and phosphosphingolipids, preferably phosphatidylcholines. The phospholipid can also be a soy lecithin.

[0006] In certain embodiments, the phospholipid is present, in the composition for use in (or used in) preparing the rhamnosome or rhamnolipid modified liposome, or in the final rhamnosome or rhamnolipid modified liposome composition, in an amount of about 1 % to about 99% by weight, about 5% to about 99% by weight, about 10% to about 99% by weight, about 20% to about 99% by weight, about 30% to about 99% by weight, about 40% to about 99% by weight, about 50% to about 99% by weight, about 60% to about 99% by weight, about 70% to about 99% by weight, about 80% to about 99% by weight, about 1 % to about 90% by weight, about 1 % to about 80% by weight, about 1 % to about 70% by weight, about 1 % to about 60% by weight, about 1 % to about 50% by weight, about 1 % to about 40% by weight, about 1 % to about 30% by weight, about 1 % to about 20% by weight, about 1% to about 10% by weight, about 10% to about 90% by weight, about 20% to about 80% by weight, about 30% to about 70% by weight, about 40% to about 60% by weight, based on the total weight of the composition.

[0007] The rhamnosomes and rhamnolipid modified liposomes disclosed herein can also comprise at least one sterol. Examples of preferred sterols include cholesterol, 7-dehydrocholesterol, potassium cholesterol sulfate, cholesteryl succinate, 25-hydroxy- 7-dehydrocholesterol, ergosterol, fucosterol, hopanoids, hydroxysteroid, phytosterols likefor example campesterol, sitosterol, and stigmasterol, steroids and zoosterols, with cholesterol being especially preferred.

[0008] The rhamnosomes and rhamnolipid modified liposomes disclosed herein can also comprise at least one active agent, including, for example ceramides, sphingoid bases, sterols, peptides, amino acids, phenols, polyphenols, vitamins, extracts from plants or algae and cosmetic oils. The active agent can be, for example, agricultural, cosmetic, pharmaceutical and / or nutraceutical active agents.BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 provides corresponding curcumin retention (%) values for curcumin-loaded rhamnosomes with purified mono-rhamnolipid and di-rhamnolipid.

[0010] Figure 2 shows the Mean Particle Diameter for rhamnosomes prepared using the ethanol injection method and acidulated SEP-RM (A-SEP-RM) in 0.01 M Phosphate Buffer (PB) at different pHs (the day of formation, and 1 week after formation).

[0011] Figure 3 shows the Mean Particle Diameter for rhamnosomes prepared using the ethanol injection method and purified mono-rhamnolipid in 0.01 M Phosphate Buffer (PB) at different pHs (the day of formation, and 1 week after formation).

[0012] Figure 4 shows the Mean Particle Diameter for rhamnosomes prepared using the ethanol injection method and purified di-rhamnolipid in 0.01 M Phosphate Buffer (PB) at different pHs (the day of formation, and 1 week after formation).

[0013] Figure 5 shows the Mean Particle Diameter for rhamnosomes prepared using the ethanol injection method and Na-SEP-RM in 0.01 M Phosphate Buffer (PB) at different pHs (the day of formation, and 1 week after formation).

[0014] Figure 6 shows the Mean Particle Diameter for rhamnosomes prepared using an aqueous solution injection method and Na-SEP-RM in 0.01 M Phosphate Buffer (PB) at different pHs (the day of formation, and 1 week after formation).

[0015] Figure 7 shows the Mean Particle Diameter for rhamnosomes prepared using the ethanol injection method (unbuffered (i.e., injection into H2O without any buffer or salt), 150mM aqueous NaCI (i.e., injection is into aqueous salt without any phosphatebuffer), or 0.01 M phosphate buffer at various pH values) and acidulated SEP-RM (A- SEP-RM) rhamnolipid (measured the day of formation, and 1 week after formation).

[0016] Figure 8 shows the PDI for rhamnosomes prepared using the ethanol injection method (unbuffered, 150mM aqueous NaCI, or 0.01 M phosphate buffer at various pH values) and acidulated SEP-RM (A-SEP-RM) rhamnolipid (measured the day of formation, and 1 week after formation).

[0017] Figure 9 shows the Mean Particle Diameter for liposomes prepared using the ethanol injection method (unbuffered, 150mM aqueous NaCI, or 0.01 M phosphate buffer at various pH values) and Soy Phosphatidylcholine (PC) (measured the day of formation, and 1 week after formation).

[0018] Figure 10 shows the PDI for liposomes prepared using the ethanol injection method (unbuffered, 150mM aqueous NaCI, or 0.01 M phosphate buffer at various pH values) and Soy PC (measured the day of formation, and 1 week after formation).

[0019] Figure 11 shows the Mean Particle Diameter for acidulated SEP-RM (A- SEP-RM) rhamnolipid (RL) modified liposomes prepared using the ethanol injection method (unbuffered, 150mM aqueous NaCI, or 0.01 M phosphate buffer at various pH values) and 3:1 RL / Soy PC (measured the day of formation, and 1 week after formation).

[0020] Figure 12 shows the PDI for acidulated SEP-RM (A-SEP-RM) rhamnolipid (RL) modified liposomes prepared using the ethanol injection method (unbuffered, 150mM aqueous NaCI, or 0.01 M phosphate buffer at various pH values) and 3:1 RL / Soy PC (measured the day of formation, and 1 week after formation).

[0021] Figure 13 shows the Mean Particle Diameter for acidulated SEP-RM (A- SEP-RM) rhamnolipid (RL) modified liposomes prepared using the ethanol injection method (unbuffered, 150mM aqueous NaCI, or 0.01 M phosphate buffer at various pH values) and 1 :1 RL / Soy PC (measured the day of formation, and 1 week after formation).

[0022] Figure 14 shows the PDI for acidulated SEP-RM (A-SEP-RM) rhamnolipid (RL) modified liposomes prepared using the ethanol injection method (unbuffered, 150mM aqueous NaCI, or 0.01 M phosphate buffer at various pH values) and 1 :1 RL / Soy PC (measured the day of formation, and 1 week after formation).

[0023] Figure 15 shows the Mean Particle Diameter for acidulated SEP-RM (A- SEP-RM) rhamnolipid (RL) modified liposomes prepared using the ethanol injection method (unbuffered, 150mM aqueous NaCI, or 0.01 M phosphate buffer at various pH values) and 1 :3 RL / Soy PC (measured the day of formation, and 1 week after formation).

[0024] Figure 16 shows the PDI for acidulated SEP-RM (A-SEP-RM) rhamnolipid (RL) modified liposomes prepared using the ethanol injection method (unbuffered, 150mM aqueous NaCI, or 0.01 M phosphate buffer at various pH values) and 1 :3 RL / Soy PC (measured the day of formation, and 1 week after formation).

[0025] Figure 17 shows the Mean Particle Diameter for acidulated SEP-RM (A- SEP-RM) rhamnolipid (RL) modified liposomes prepared using the ethanol injection method (unbuffered, 150mM aqueous NaCI, or 0.01 M phosphate buffer at various pH values) and 1 :9 RL / Soy PC (measured the day of formation, and 1 week after formation).

[0026] Figure 18 shows the PDI for acidulated SEP-RM (A-SEP-RM) rhamnolipid (RL) modified liposomes prepared using the ethanol injection method (unbuffered, 150mM aqueous NaCI, or 0.01 M phosphate buffer at various pH values) and 1 :9 RL / Soy PC (measured the day of formation, and 1 week after formation).

[0027] Figure 19 shows the Mean Particle Diameter for acidulated SEP-RM (A- SEP-RM) rhamnolipid (RL) modified liposomes prepared using the ethanol injection method (unbuffered, 150mM aqueous NaCI, or 0.01 M phosphate buffer at various pH values) and 1 :19 RL / Soy PC (measured the day of formation, and 1 week after formation).

[0028] Figure 20 shows the PDI for acidulated SEP-RM (A-SEP-RM) rhamnolipid (RL) modified liposomes prepared using the ethanol injection method (unbuffered, 150mM aqueous NaCI, or 0.01 M phosphate buffer at various pH values) and 1 :19 RL / Soy PC (measured the day of formation, and 1 week after formation).

[0029] Figure 21 shows the Mean Particle Diameter for rhamnosomes prepared using the ethanol injection method (unbuffered H2O) and acidulated SEP-RM (A-SEP- RM) rhamnolipid together with various amounts of cholesterol (measured the day of formation, and 1 week after formation).

[0030] Figure 22 shows the PDI for rhamnosomes prepared using the ethanol injection method (unbuffered H2O) and acidulated SEP-RM (A-SEP-RM) rhamnolipid together with various amounts of cholesterol (measured the day of formation, and 1 week after formation).

[0031] Figure 23 shows the Mean Particle Diameter for rhamnosomes prepared using the ethanol injection method (unbuffered, 150mM aqueous NaCI, or 0.01 M phosphate buffer at various pH values) and acidulated SEP-RM (A-SEP-RM) rhamnolipid (RL) in the presence of cholesterol (80:20 RL / cholesterol).

[0032] Figure 24 shows the PDI for rhamnosomes prepared using the ethanol injection method (unbuffered, 150mM aqueous NaCI, or 0.01 M phosphate buffer at various pH values) and acidulated SEP-RM (A-SEP-RM) rhamnolipid (RL) in the presence of cholesterol (80:20 RL / cholesterol).

[0033] Figure 25 shows the Mean Particle Diameter for rhamnosomes prepared (unbuffered, 150mM aqueous NaCI, or 0.01 M phosphate buffer at various pH values) in the presence of 0 wt% Curcumin (measured the day of formation, and 1 week after formation (stored at room temperature).

[0034] Figure 26 shows the Mean Particle Diameter for rhamnosomes prepared (unbuffered, 150mM aqueous NaCI, or 0.01 M phosphate buffer at various pH values) in the presence of 5 wt% Curcumin (measured the day of formation, and 1 week after formation (stored at room temperature, and 10°C)).

[0035] Figure 27 shows the Mean Particle Diameter for rhamnosomes prepared (unbuffered, 150mM aqueous NaCI, or 0.01 M phosphate buffer at various pH values) in the presence of 15 wt% Curcumin (measured the day of formation, and 1 week after formation (stored at room temperature, and 10°C)).

[0036] Figure 28 shows the Mean Particle Diameter for purified di-rhamnolipid (Di- RL) rhamnosomes prepared using the ethanol injection method (unbuffered, 150mM aqueous NaCI, or 0.01 M phosphate buffer at various pH values) and Di-RL (measured the day of formation, and 1 week after formation).

[0037] Figure 29 shows the PDI for purified di-rhamnolipid (Di-RL) rhamnosomes prepared using the ethanol injection method (unbuffered, 150mM aqueous NaCI, or 0.01 M phosphate buffer at various pH values) and Di-RL (measured the day of formation, and 1 week after formation).

[0038] Figure 30 shows the Mean Particle Diameter for Soy PC liposomes prepared using the ethanol injection method (unbuffered, 150mM aqueous NaCI, or 0.01 M phosphate buffer at various pH values) and Soy PC (measured the day of formation, and 1 week after formation).

[0039] Figure 31 shows the PDI for Soy PC liposomes prepared using the ethanol injection method (unbuffered, 150mM aqueous NaCI, or 0.01 M phosphate buffer at various pH values) and Soy PC (measured the day of formation, and 1 week after formation).

[0040] Figure 32 shows the Mean Particle Diameter for purified di-rhamnolipid (Di- RL) modified Soy PC liposomes prepared using the ethanol injection method (unbuffered, 150mM aqueous NaCI, or 0.01 M phosphate buffer at various pH values) and a 1 :3 Di- RL / Soy PC ratio (measured the day of formation, and 1 week after formation).

[0041] Figure 33 shows the PDI for purified di-rhamnolipid (Di-RL) modified Soy PC liposomes prepared using the ethanol injection method (unbuffered, 150mM aqueous NaCI, or 0.01 M phosphate buffer at various pH values) and a 1 :3 Di-RL / Soy PC ratio (measured the day of formation, and 1 week after formation).

[0042] Figures 34 A-B compare the Mean Particle Diameter of acidulated SEP- RM (A-SEP-RM) rhamnolipid modified Soy PC liposomes prepared using the ethanol injection method (unbuffered, 150mM aqueous NaCI, or 0.01 M phosphate buffer at various pH values) and a 1 :3 acidulated SEP-RM (A-SEP-RM) RL / Soy PC ratio, versus purified di-rhamnolipid (Di-RL) modified Soy PC liposomes prepared using the ethanol injection method (unbuffered, 150mM aqueous NaCI, or 0.01 M phosphate buffer at various pH values) and a 1 :3 Di-RL / Soy PC ratio (measured the day of formation, and 1 week after formation).

[0043] Figures 35 A-B compares the PDI of acidulated SEP-RM (A-SEP-RM) rhamnolipid modified Soy PC liposomes prepared using the ethanol injection method (unbuffered, 150mM aqueous NaCI, or 0.01 M phosphate buffer at various pH values) and a 1 :3 acidulated SEP-RM (A-SEP-RM) RL / Soy PC ratio, versus purified di-rhamnolipid (Di-RL) modified Soy PC liposomes prepared using the ethanol injection method (unbuffered, 150mM aqueous NaCI, or 0.01 M phosphate buffer at various pH values) and a 1 :3 Di-RL / Soy PC ratio (measured the day of formation, and 1 week after formation).

[0044] Figures 36 A-B show the Mean Particle Diameter (Figure 36 A) and PDI (Figure 36 B) of liposomes prepared to assess the fabrication of mixed purified di- rhamnolipid / Soy PC liposomes in the presence 0.01 M Phosphate Buffer (pH 5.0) using the ethanol injection method (measured the day of formation, and 1 week after formation).

[0045] Figures 37 A-B show the Mean Particle Diameter (Figure 37 A) and PDI (Figure 37 B) of liposomes prepared to assess the effect of cholesterol on the formation of rhamnosomes prepared using the ethanol injection method and purified di-rhamnolipid (unbuffered, 150mM aqueous NaCI, or 0.01 M phosphate buffer at various pH values) (measured the day of formation, and 1 week after formation).

[0046] Figures 38 A-B shows the initial mean particle diameter (Figure 38 A) and PDI (Figure 38 B) of rhamnosomes fabricated without active and with increasing loading levels of curcumin (fabricated from unbuffered H2O or 0.01 M phosphate buffer at various pH values).

[0047] Figures 39 A-B shows the change in mean particle diameter (Figure 39 A) and PDI (Figure 39 B) of rhamnosomes fabricated with 5 wt% curcumin on lipid (fabricated from unbuffered H2O or 0.01 M phosphate buffer at various pH values) with time upon storage at ambient temperature (RT) or 10QC.

[0048] Figure 40 shows the change in encapsulated curcumin for rhamnosomes fabricated with 5 wt% curcumin on lipid (fabricated from unbuffered H2O or 0.01 M phosphate buffer at various pH values) with time upon storage at ambient temperature (RT) or 10QC.

[0049] Figures 41 A-B shows the change in mean particle diameter (Figure 41 A) and PDI (Figure 41 B) of rhamnosomes fabricated with 10 wt% curcumin on lipid (fabricated from unbuffered H2O or 0.01 M phosphate buffer at various pH values) with time upon storage at ambient temperature (RT) or 10QC.

[0050] Figure 42 shows the change in encapsulated curcumin for rhamnosomes fabricated with 10 wt% curcumin on lipid (fabricated from unbuffered H2O or 0.01 M phosphate buffer at various pH values) with time upon storage at ambient temperature (RT) or 10QC.

[0051] Figures 43 A-B shows the change in encapsulated curcumin for rhamnosomes fabricated with increasing loading levels of curcumin on lipid (fabricated from unbuffered H2O (Figure 43 A) or 0.01 M phosphate buffer at pH 6.0 (Figure 43 B)) with time upon storage at ambient temperature (RT) or 10SC.

[0052] Figures 44 A-B shows the change in encapsulated curcumin for soy phosphatidylcholine liposomes fabricated with increasing loading levels of curcumin on lipid (fabricated from H3PO4-adjusted H2O (Figure 44 A) or 0.01 M phosphate buffer at pH 6.0 (Figure 44 B)) with time upon storage at ambient temperature (RT) or 10SC.

[0053] Figures 45 A-B shows the change in encapsulated curcumin for DPPC liposomes fabricated with increasing loading levels of curcumin on lipid (fabricated from HsPC -adjusted H2O (Figure 45 A) or 0.01 M phosphate buffer at pH 6.0 (Figure 45 B)) with time upon storage at ambient temperature (RT) or 10QC.

[0054] Figure 46 shows the change in encapsulated curcumin for rhamnosomes prepared from Na-SEP-RM rhamnolipid (RL) and pH 3.0 buffer, fabricated with increasing loading levels of curcumin on lipid, with time upon storage at ambient temperature (RT) or 10QC.

[0055] Figure 47 shows the change in encapsulated curcumin for rhamnosomes prepared from purified mono-RL and pH 5.5 buffer, fabricated with increasing loading levels of curcumin on lipid, with time upon storage at ambient temperature (RT) or 10QC.

[0056] Figure 48 shows the change in encapsulated curcumin for rhamnosomes prepared from purified di-RL and pH 5.5 buffer, fabricated with increasing loading levels of curcumin on lipid, with time upon storage at ambient temperature (RT) or 10QC.DETAILED DESCRIPTION OF THE INVENTION

[0057] “Biorenewable Carbon Index” (BCI) refers to a calculation of the percent carbon derived from a biorenewable resource, and is calculated based on the number of biorenewable carbons divided by the total number of carbons in the entire molecule.

[0058] “Biorenewable” is defined herein as originating from animal, plant, or marine material.

[0059] The term liposome is generally accepted in the art as including lipid vesicles (e.g., wherein the lipid content comprises phospholipids). The term rhamnolipid modified liposome (or rhamnolipid containing liposome) is preferably understood to mean a liposome comprising rhamnolipid. In some embodiments, the term rhamnolipid modified liposome (or rhamnolipid containing liposome) is a liposome comprising rhamnolipid in an amount less than 50 wt% of the total lipid content. The term rhamnosome can be interchangeable with the term rhamnolipid modified liposome (or rhamnolipid containing liposome). As used herein, the term rhamnosome is preferably understood to mean a liposome comprising rhamnolipid, preferably in an amount equal to or greater than 50 wt% rhamnolipid.

[0060] As defined herein, a “rhamnolipid” is a glycolipid that has a lipid portion that includes one or more, typically linear, saturated or unsaturated [3-hydroxy-carboxylic acid moieties and a saccharide portion of one or more units of rhamnose.

[0061] The saccharide portion and the lipid portion are linked via a [3-glycosidic bond between the 1 -OH group of a rhamnose moiety of the saccharide portion and the 3- OH group of a [3-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 ,4 [3-glycosidic bond. In embodiments where two or more [3-hydroxy-carboxylic acids are present in a rhamnolipid,the [3-hydroxy-carboxylic acid moieties are selected independently from each other. [3- hydroxy carboxylic acid moieties may in some embodiments be identical. In some embodiments, they are different from each other.

[0062] The present technology generally relates to liposomal compositions that comprise a particular mixture of rhamnolipids. The rhamnolipids in the mixture of rhamnolipids of the present technology 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)— -CH2- 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.

[0063] 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 otherthan 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, thio and sulfonyl.

[0064] In a more particular embodiment, the rhamnolipids or rhamnolipid salts in said structure has 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 silver or alkali metals Li, Na, or K, alkali earth metals Mg or Ca, or transition metals Mn, Fe, Cu, or Zn. In the cases of the alkali earth and transition metals, multiple rhamnolipid salt moieties may associate with each metal

[0065] The mixture of rhamnolipids comprises a mixture of mono-rhamnolipids and di-rhamnolipids. In other embodiments, the rhamnolipid composition comprises purified mono-rhamnolipids alone. In further embodiments, the rhamnolipid composition comprises purified di-rhamnolipids alone.

[0066] In some embodiments, the mono-rhamnolipids may be present in an amount of about 5% to 95%, alternatively about 10% to 90%, alternatively about 15% to 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 15% to about 80%, alternatively about 20% to about 80%, alternatively about 30% to about 80%, alternatively about 40% to about 80%, alternatively about 30% to about 70%, alternatively about 30% to about 60%, alternatively about 30% to about 50%, based on the total weight of rhamnolipids and as measured by high-performance liquid chromatography (HPLC), 1 H NMR and / or reverse phase UPLC-MS.

[0067] In some embodiments, the mono-rhamnolipids may be present in an amount of about 10% to about 47%, alternatively about 15% to about 45%, alternatively about 20% to about 45%, alternatively about 30% to about 45%, alternatively about 40% to about 45%, alternatively about 43% to about 45%, 10% to about 48%, alternatively about 15% to about 48%, alternatively about 20% to about 48%, alternatively about 30% to about 48%, alternatively about 40% to about 48%, alternatively about 43% to about 48% alternatively about 10% to about 47%, alternatively about 15% to about 47%, alternatively about 20% to about 47%, alternatively about 30% to about 47%, alternatively about 40% to about 47%, alternatively about 43% to about 47%, based on the total weight of rhamnolipids.

[0068] The di-rhamnolipids may be present in an amount of about 52% to about 90% by weight, alternatively about 52% to about 85%, alternatively about 52% to about 80%, alternatively about 52% to about 70%, alternatively about 52% to about 60%, alternatively about 52% to about 57%, alternatively, about 53% to about 90% by weight, alternatively about 53% to about 85%, alternatively about 53% to about 80%, alternatively about 53% to about 70%, alternatively about 53% to about 60%, alternatively about 53% to about 57%, alternatively about 54% to about 85%, alternatively about 55% to about 80%, alternatively about 55% to about 70%, alternatively about 55% to about 60%, alternatively about 55% to about 57% by weight, based on the total weight of rhamnolipids and as measured by high-performance liquid chromatography (HPLC), 1 H NMR and / or reverse phase UPLC-MS. The ratio of mono-rhamnolipids:di-rhamnolipids can be from about 10:90 to about 48:52, alternatively about 47:53, alternatively about 50:50, alternatively about 40:60 to about 45:55. In some embodiments, the ratio of mono- rhamnolipids:di-rhamnolipids can range from 43.5:56.5 to 45:55, alternatively from 43:57 to 45:55 or from 43:57 to 48:52.

[0069] 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. Rha-C10-C10 may be present in the mixture in an amount of about 5% to 38%, alternatively about 5% to about 20%, alternatively about 5% to about 15%, alternatively about 10% to about 15%, alternatively about 10% to about 14%, alternatively about 10% to about 12%, alternatively about 10% to about 38%, alternatively about 10% to about 38%, alternatively about 15% to about 37.5%, alternatively about 20% to about 37.5%, alternatively about 25% to about 37.5%, alternatively about 29% to about 37.5%, alternatively about 30% to about 50%, alternatively about 35% to about 45%, alternatively about 35% to about 37% by weight based on the total weight of rhamnolipids, as measured by high-performance liquid chromatography (HPLC), 1 H NMR and / or reverse phase UPLC-MS. RhaRha-C10-C10 may be present in the mixture in an amount of about 1% to about 20%, alternatively about 5% to about 15%, alternatively about 8% to about 12%; alternatively about 34% 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 36% to about 38%, alternatively about 34% to about 80%, by weight based on the total weight of rhamnolipids in the mixture of rhamnolipids, as measured by high-performance liquid chromatography (HPLC), 1 H NMR and / or reverse phase UPLC-MS.

[0070] In addition to Rha-C10-C10 and RhaRha-C10-C10, the mixture of rhamnolipids may comprise RhaRha-C12-C12 in an amount of about 0.05% to about 1 %, alternatively about 0.05% to about 0.5%, alternatively about 0.1 % to about 0.6%, alternatively about 0.2% to about 0.5%, alternatively about 0.05% to about 0.2%, RhaRha-C10-C12 in an amount of about 5% to about 15%, alternatively about 4% to about 8%, alternatively about 5% to about 7%, alternatively about 9% to about 12%, alternatively about 1 % to about 5%, alternatively about 1 % to about 4%, alternatively about 1 % to about 3%, 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 0.2% to about 6% by weight, alternatively about 0.5% to about 3%, alternatively about 0.5% to about 2%, alternatively about 2% to about 5%, alternatively about 3.5% to about 5% by weight based on the total weight of rhamnolipids, each being measured by high-performance liquid chromatography (HPLC). The mixture of rhamnolipids may also comprise, for example,an amount of RhaRha-C10-C12:1 in an amount of about 0.2% to about 5% by weight, alternatively about 1 % to about 5% by weight, alternatively about 2% to about 4% by weight, alternatively about 0.2% to about 3% by weight, alternatively about 0.2% to about 2% by weight, alternatively about 0.2% to about 1% by weight, alternatively about 0.5% to about 1 % by weight, alternatively 1 % to about 4% 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 0.2% to about 0.8%, alternatively about 0.3% to about 0.6%, alternatively about 1 % to about 4%, alternatively about 1 % to about 3%, alternatively about 1 % to about 2% by weight, based on the total weight of rhamnolipids; an amount of Rha-C8-C10 in the range of about 0.2% to about 5% by weight, alternatively about 1% to about 4% by weight, alternatively about 0.2% to about 0.8%, alternatively about 0.3% to about 0.6%, alternatively about 1 % to about 4%, alternatively about 1 % to about 3%, alternatively about 1 % to about 2% based on the total weight of rhamnolipids; or any combination thereof, as measured by high-performance liquid chromatography (HPLC), 1 H NMR and / or reverse phase UPLC-MS.

[0071] In one aspect, the rhamnosomes and rhamnolipid modified liposomes disclosed herein comprise at least one phospholipid. The phospholipid can be selected from phosphatidylcholines, also known as lecithins and often abbreviated as PC, phosphatidylethanolamines, also known as cephalins and often abbreviated as PE, phosphatidic acid also known as phosphatidates and often abbreviated as PA, phosphatidylserines often abbreviated as PS, phosphoinositides, like for example phosphatidylinositol (often abbreviated as PI), phosphatidylinositol phosphate (often abbreviated as PIP), phosphatidylinositol bisphosphate (often abbreviated as PIP2) and phosphatidylinositol trisphosphate (often abbreviated as PIP3), and phosphosphingolipids, like for example ceramide phosphorylcholines (often abbreviated as SPH), ceramide phosphorylethanolamines (often abbreviated as Cer-PE) and ceramide phosphoryllipids, preferably phosphatidylcholines.

[0072] Phospholipids are commonly obtained from biological sources, for examples plants and animals. Industrial common sources are soya, rapeseed, sunflower, chicken eggs, bovine milk and fish eggs.

[0073] Preferably the rhamnosomes and / or the rhamnolipid modified liposomes according to the instant disclosure can comprises phosphatidylcholines, wherein the phosphatidylcholine is preferably derived from plants, preferably from the group of soya, sunflowers, rapeseed, lupines, pea, and beans, with soya and sunflowers being especially preferred and sunflower the most.

[0074] In a further aspect, the rhamnosomes and rhamnolipid modified liposomes disclosed herein can comprise at least one active agent. As used herein, the term active agent includes, for example, a chemical compound (or in some cases a collection of chemical compounds) that produces a desired effect or response. Active agents can include, for example, agents having utility in the agricultural, cosmetic, personal care (e.g., oral care), pharmaceutical, and / or nutraceutical fields. For example, the active agent can be ceramides, sphingoid bases, sterols, peptides, amino acids, phenols, polyphenols, vitamins, extracts from plants or algae and cosmetic oils, or combinations thereof. The active can also include nucleic acids (e.g., RNA, DNA, antisense oligonucleotides, small interfering RNAs, aptamers, ribozymes, and modified derivatives thereof).

[0075] In a still further aspect, the rhamnosomes and rhamnolipid modified liposomes disclosed herein can also comprise at least one sterol. Preferred sterols include, for example, cholesterol, 7-dehydrocholesterol, potassium cholesterol sulfate, cholesteryl succinate, 25-hydroxy-7-dehydrocholesterol, ergosterol, fucosterol, hopanoids, hydroxysteroid, phytosterols like for example campesterol, sitosterol, and stigmasterol, steroids and zoosterols, with cholesterol being especially preferred.

[0076] In a still further aspect, the rhamnosomes and rhamnolipid modified liposomes disclosed herein can also comprise additional formulation components that can modify properties such as phase transition temperature, elasticity, deformability, and skin and / or plant cuticle penetration abilities. For example, so-called “edge activators”, including surfactants, polyols such as glycerin or propylene glycol, and other additives known to those skilled in the art may be used.

[0077] Rhamnosomes and rhamnolipid containing liposomes of the present disclosure can be fabricated in accordance with standard methods of preparation.Standard methods of preparation include, for example, those methods described in the following: (1 ) “Emerging Applications of Nanoparticles and Architectural Nanostructures, Chapter 15: Contemporary industrial practice for manufacturing of nanomedicines” (by S. Tinkov) (pp. 447-500 (2018)); (2) Liu P, Chen G, Zhang J. A Review of Liposomes as a Drug Delivery System: Current Status of Approved Products, Regulatory Environments, and Future Perspectives. Molecules. 2022 Feb 17;27(4):1372; or (3) Nsairat H, Khater D, Sayed U, Odeh F, Al Bawab A, Alshaer W. Liposomes: structure, composition, types, and clinical applications. Heliyon. 2022 May 13;8(5):e09394. The contents of these references are hereby incorporated by reference in their entirety.

[0078] In one aspect, the rhamnolipid containing liposomes of the present disclosure can be prepared using a thin film hydration method. This method comprises a first film casting step, wherein all lipids, and optionally an active (e.g., hydrophobic active agent or drug), are dissolved in a suitable organic solvent using a round-bottom flask. The organic solvent is then evaporated gently under reduced pressure to create, or cast, a thin film layer on the bottom of the round-bottom flask. During a subsequent hydration step, the thin film is then hydrated (e.g., at above the transition temperature (Tm) of the used lipid) with an aqueous buffer solution. The hydration solution may also contain the active (e.g. hydrophilic drugs) to be loaded into the rhamnosomes, liposomes, or rhamnolipid modified liposome aqueous core. After hydration, an optional extrusion step involves forcing liposome suspensions through membranes with specific pore sizes, often performed above the lipid phase-transition temperature. This results in liposomes size reduction with narrow size distributions. Liposomes resizing, lamellarity types and particles distributions can be controlled by either extrusion through membranes (e.g., polycarbonate membranes) of specific pore sizes or the use of bath or probe sonicators. Extrusion can include multiple membrane extrusion steps (e.g., three extrusion steps - 400, 200, and 100nm pore sizes). In other embodiments, particle characteristics (e.g., particle size) can be refined through repeated freeze I thaw cycles.

[0079] After formation of the rhamnosome or rhamnolipid containing liposomes, substances (e.g., unencapsulated actives and contaminants) often remain in the surrounding liquid. A key step post-production may involve separation (e.g., removing any leftover organic solvents used in the process (e.g., volatiles stripping process), orremoving any leftover actives or other impurities). This step can include active separation methods. For example, Sephadex size exclusion can be used (e.g., Millipore Sigma’s PD midiTrap G-25 centrifuge columns). Dialysis size exclusion can also be used. This step can also comprise passive separation methods (i.e., gravimetric settling).

[0080] In another aspect, the rhamnosome or rhamnolipid containing liposomes of the present disclosure can be prepared using solvent injection methods. These injection methods can be classified according to the type of organic solvent used (e.g., ethanol). For example, the ethanol injection method (i.e., ethosome method) involves injection of an ethanolic solution comprising one or more lipids into a larger volume of aqueous phase, which leads to rapid formation of vesicles without passing through any intermediate process. Upon injection, the unfavorable exposure of lipids to the aqueous medium results in arrangement and precipitation of lipids at the boundary phase between ethanol and water in the form of bilayer lipid fragments. During the ethanol injection method, factors such as mixing speed, injection velocity, pH, lipid concentration, temperature and osmolarity, can substantially influence the features of the produced liposomes.

[0081] In some instances, the solvent injection method utilizes an aqueous solution of one or more lipids instead of an organic solvent-based solution. In these instances, one or more lipids are soluble in water, or are solubilized in water, for example as a micellar solution. In one preferred aspect, the composition of the aqueous solution comprises a water-soluble carboxylate salt of rhamnolipid. In this aspect, the pH of the aqueous solution is sufficiently high to ensure solubility of the rhamnolipid. In some instances, the aqueous solution further comprises a water-soluble active. In other instances, the aqueous solution further comprises components that are successfully solubilized into water by the presence of the water-soluble rhamnolipid and that are otherwise insoluble, marginally soluble, or poorly soluble in water on their own, for example water-insoluble phospholipids, sterols such as cholesterol, and water-insoluble actives. Optionally, the aqueous solution may further comprise alcohols, polyols, and / or surfactants, provided that these components are water soluble or are solubilized into the aqueous solution. The aqueous lipid solution is injected into a second aqueous phase that is at a lower pH, which leads to rapid formation of vesicles.

[0082] In some instances, the solvent injection method may be conducted in a continuous fabrication process that utilizes a flow reactor, wherein a flowing stream of solvent, aqueous solution, or aqueous buffer comprising one or more lipids is mixed with a flowing stream of an aqueous phase. A non-limiting example of such a flow reactor is a microfluidic device. In some instances, the method is preferably performed without subsequent extrusion, and with passive separation.

[0083] In another aspect, the rhamnosome or rhamnolipid containing liposomes of the present disclosure can be prepared using inverse aqueous injection methods. These methods involve the injection of an acid into an aqueous solution comprising rhamnolipid (receiving solution). Non-limiting examples of suitable acids include sulfuric, phosphoric, hydrochloric, nitric, formic, acetic, propionic, citric, oxalic, carbonic, lactic, and glycolic acid. Additional examples of suitable acids include alkanoic acids of the formula RCOOH, wherein R is aliphatic or aromatic, linear or branched, acyclic or cyclic, saturated or unsaturated, and with a total number of carbons from 4 to 22. In some instances, the acid is in the form of an aqueous solution.

[0084] Prior to acid injection, the receiving solution in the inverse aqueous injection method is preferably at a pH sufficient to provide a homogenous solution of rhamnolipid free of precipitate, for example as a micellar solution. In some aspects, the pH of the receiving solution prior to acid injection is in the range of 5-11 , alternatively 5.5-10, alternatively 6-9. In one preferred aspect, the receiving solution comprises a water- soluble carboxylate salt of rhamnolipid. The receiving solution may further comprise one or more water-soluble active agents. Optionally, the receiving solution may comprise components that are successfully solubilized into water by the presence of the water- soluble rhamnolipid and that are otherwise insoluble, marginally soluble, or poorly soluble in water on their own, for example water-insoluble phospholipids, sterols such as cholesterol, and water-insoluble actives.

[0085] In the inverse aqueous injection method, acid is injected in amount necessary to result in controlled formation of vesicles and bilayer structures without precipitation or phase separation of oily or gummy acidulated rhamnolipids or rhamnolipid-comprising insoluble material. Preferably, the pH of the resulting fluid is inthe range of 3-5, alternatively 4-5. The inverse aqueous injection method can enable the preparation of lipid fluids with mean particle sizes of less than about 500 nm and at concentrations that are otherwise difficult to achieve without the use of high-energy input methods such as high-speed homogenization or micro-fluidization.

[0086] Numerous parameters are affected by the compositions and methods employed to fabricate the rhamnolipid containing liposomes described herein. For example, parameters such as particle size, polydispersity, surface charge, vesicle lamellarity, elasticity, deformability, actives (e.g., drug) loading, and encapsulation efficiency can be used to characterize the rhamnolipid containing liposomes described herein.

[0087] Depending on the composition and method of preparation, rhamnolipid containing liposomes described herein can vary in size distribution, either forming, for example, uniform small size vesicles or a heterogeneous mixture of small to large vesicles. Determination of particle size distribution can be performed using a number of techniques, including for example dynamic light scattering, also sometimes referred to as photon correlation spectroscopy, flow cytometry, or microscopy techniques such as cryo- TEM. In certain aspects, the rhamnolipid containing liposomes described herein have a mean particle size of from 15 nm to 800 nm, from 50 nm to 500 nm, from 60 nm to 350 nm or from 80 nm to 240 nm.

[0088] With respect to characterization of particle size distribution, “polydispersity index” (PDI) is a parameter that can be used to define the size range of rhamnolipid containing liposomes described herein. The term “polydispersity” (or “dispersity” as recommended by IUPAC) is used to describe the degree of non-uniformity of a size distribution of particles. In certain aspects, the rhamnolipid containing liposomes described herein have a PDI of from 0.05 to 0.9, from 0.05 to 0.7, from 0.05 to 0.6, from 0.05 to 0.5, from 0.05 to 0.4, from 0.05 to 0.3, from 0.05 to 0.2, or from 0.05 to 0.1 .

[0089] In other aspects, rhamnosomes or rhamnolipid containing liposomes described herein can be characterized with respect to actives loading capacity and / or actives encapsulation or loading efficiency. Actives loading can be attained either passively (i.e., the active is encapsulated during rhamnosome or rhamnolipid containingliposome formation) or actively (i.e., after rhamnosome or rhamnolipid containing liposome formation). Actives loading is the wt.% of active incorporated in a rhamnosome or liposome on the basis of the total mass of active, lipids, sterols, and other rhamnosome or rhamnolipid containing liposome organic components. Actives loading capacity (LC%) refers to the maximum amount of active that can be loaded while maintaining successful fabrication and meeting one or more stability criteria. In one non-limiting aspect, being free of visual precipitation or sedimentation after maintaining a fabricated sample overnight at 10SC can be used as a stability criterium. LC% can be calculated as the weight of the active (e.g., drug) encapsulated in the rhamnosome or rhamnolipid containing liposomes divided by the total weight of the rhamnosome or rhamnolipid containing liposomes x 100. In certain aspects, the rhamnosome or rhamnolipid containing liposomes described herein can have an actives loading capacity of 2-50%, alternatively 5-30%, alternatively 10-20%.

[0090] Actives encapsulation or loading efficiency (LE%) can be calculated as (total active (e.g., drug) added - free non-entrapped active) divided by the total active added x 100. In certain aspects, the rhamnosomes or rhamnolipid containing liposomes described herein can have an actives loading efficiency of 30-100%, alternatively 50- 100%, alternatively 70-100%, alternatively 90-100%, alternatively > 50%, alternatively > 60%, alternatively > 70%, alternatively > 80%, alternatively > 90%, or alternatively > 95%.

[0091] In another aspect, rhamnosomes or rhamnolipid containing liposomes described herein can be characterized with respect to stability. Parameters used to characterize stability include, without limitation: retention of active (e.g., change in supernatant active recovery); change in particle size (e.g., change in mean particle diameter and / or change in PDI); flocculation I sedimentation (e.g., visual assessment and / or change in supernatant lipid recovery); and chemical stability (e.g., HPLC and / or Mass Spectrometry analysis).

[0092] In another aspect, rhamnosomes and rhamnolipid-modified liposomes described herein can be characterized with respect to release of active to surrounding media and / or to a targeted substrate. Release profiles can be quantified in terms of kinetic rates of release or deposition, quantities deposited to a substrate for a given time in termsof mass per unit area or unit volume, and depth of penetration into substrates such as human skin consisting of the epidermis, dermis or hypodermis or into the plant leaf structures such as the cuticle, upper dermis, mesophyll or stoma. .Such profiles can be characterized by a number of techniques, including application of diffusion cells such as static flow cell or Franz cells, continuous flow or open-cell analysis, dynamic dialysis, fluorescence de-quenching, and microscopy techniques such as confocal fluorescence microscopy. Other techniques such as radiolabeling have also been utilized for quantitative analysis.EXAMPLES

[0093] The presently described technology and its advantages will be better understood by reference to the following examples. These examples are provided to describe specific embodiments of the present technology. By providing these examples, the inventors do not limit the scope and spirit of the present technology.Experimental ProceduresRhamnolipid Compositions

[0094] 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 Actinobacteria, Firmicutes, 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.

[0095] 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. The rhamnolipids in the mixture of rhamnolipids used in thepresent technology are separated from the fermentation medium, washed, deodorized, decolorized, and neutralized to form the purified rhamnolipid salts.

[0096] Fermentation of the whole broth then undergoes multiple purification steps. The specific combination of purification steps to obtain the rhamnolipids actives from fermentation whole broth 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 Solvent Extraction Purified Rhamnolipid Mixture (or composition), in accordance with the current specification.

[0097] For example, the SEP-RM (or Na-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) carbon treatment; (10) filtration; (11 ) solvent removal; (12) neutralization and dilution; and (13) 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.

[0098] For the acidulated SEP-RM (herein referred to as A-SEP-RM), an additional acidulation step (14) is required after the final polishing step (13). To a separatory funnel, SEP-RM (Na-SEP-RM) was added, and the pH was adjusted to acidic pH (2-3) with 1 N aq. HCI solution until acidulated SEP-RM (A-SEP-RM) crashed out of the solution. Subsequently, to the mixture in the separatory funnel was added chloroform (CHCh) and the acidulated SEP-RM (A-SEP-RM) was extracted three times with chloroform and the organic layer was collected. Anhydrous sodium sulfate was added to the organic layer to remove residual water and the entire solution was filtered using a funnel having a cotton plug. The chloroform was removed using rotary evaporator to afford acidulated SEP-RM (A-SEP-RM) as the final product.

[0099] In some embodiments, rhamnolipid can be used after a first washing step (i.e., step 7 above), without any additional processing steps (including those listed above). In these embodiments, the rhamnolipid is referred to as BWAR rhamnolipid (i.e., Bleached / Washed / Acidulated Rhamnolipid).

[0100] 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.

[0101] 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 is concentrated (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).

[0102] 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. As discussed above, to prepare acidulated SEP-RM (A- SEP-RM), an additional acidulation step is required after the polishing step.Mono-Rhamnolipid:Di-Rhamnolipid Ratio and Congener Distribution

[0103] The mono-rhamnolipid:di-rhamnolipid ratio, and congener distribution for rhamnolipid compositions of the present disclosure can be determined using UPLC-MS (Ultra-Performance Liquid Chromatography-Mass Spectrometry) and 1 H NMR (proton Nuclear Magnetic Resonance) spectroscopy. As understood by the person of ordinary skill in the art, reverse phase UPLC-MS will separate rhamnolipid congeners by chain length and relative amount of rhamnose moieties (with the more polar congeners eluting first). As understood by the person of ordinary skill in the art, 1 H NMR can determine the weight percent of mono-rhamnolipid and di-rhamnolipid congeners using maleic acid as the internal standard. The weight percent of mono-rhamnolipid actives determined by 1 H NMR can be multiplied by the relative percent areas of mono-rhamnolipid congeners determined using UPLC-MS to estimate the respective mono-rhamnolipid congener weight percents. The weight percent of di-rhamnolipid actives determined by 1 H NMR can be multiplied by the relative percent areas of di-rhamnolipid congeners determined using UPLC-MS to estimate the respective di-rhamnolipid congener weight percents.

[0104] The UPLC-MS and 1 H NMR congener distribution for rhamnolipid compositions of the present technology may comprise a distribution of congeners as follows, wherein all percentages are by relative weight based on the total weight of congeners in the composition, normalized to 100%: a) mono-rhamnolipid C8-C10 congener in an amount of about 2% to about 5%. b) di-rhamnolipid C8-C10 congener in an amount of about 2% to about 5%. c) mono-rhamnolipid C10-C10 congener in an amount of about 29% to about 42%, preferably about 35% to about 40%. d) di-rhamnolipid C10-C10 congener in an amount of about 35% to about 50%, preferably about 35% to about 45% by relative percent weight. e) mono-rhamnolipid C10-C12 congener in an amount of about 2% to about 6% by relative percent weight. f) di-rhamnolipid C10-C12 congener in an amount of about 2% to about 8% by relative percent weight.Within this technology, the total mono-rhamnolipid congeners are present in an amount ranging from approximately 39% to 48% by relative weight, and the total di-rhamnolipid congeners are present in an amount ranging from approximately 52% to 60% by relative weight.

[0105] The congener distribution for exemplary rhamnolipid compositions of the present disclosure (determined using techniques known to those skilled in the art, including 1 H NMR and reverse phase UPLC-MS), is presented below:Phospholipids

[0106] In the examples hereafter, where used, dipalmitoylphosphatidylcholine (DPPC) was >99% purity, 25 mg / mL in chloroform from Avanti Polar Lipids; purified soy phosphatidylcholine (soy PC) was >95% purity from Avanti Polar Lipids; and powdered soy lecithin was 90% purity from Alfa Aesar.Buffers

[0107] In all cases, unless otherwise indicated, the pH values listed in the Figures and tables are the initial pH values of aqueous buffers used in fabrication of rhamnosomes and rhamnosome containing I modified liposomes. The pH of the resultant aqueous compositions was subject to shifting in value depending on the lipid being used. Unbuffered water was not pH-adjusted unless otherwise indicated. Phosphate buffer saline (PBS) comprised approximately 10 mM phosphate salts and about 140 mM Na / K chloride salts. 0.01 M phosphate buffers (no additional salt) were prepared from combinations of H3PO4 and Na2HPO4.Preparation of Divalent Cation Salts of Rhamnolipid

[0108] In separate reactions, salts of Zn2+, Cu2+, Mn2+, Mg2+, and Ca2+ were prepared from SEP-RM rhamnolipid and metal2+ chloride salts (M2+CI2). In a typical procedure, rhamnolipid and M2+CI2 were combined in water and stirred for 24 hr at ambient temperature. The precipitated rhamnolipid salt was then isolated by filtration and or centrifugation, washing with H2O, and dried to recover a powder. In the case of Mg2+, precipitation was not observed and the rhamnolipid salt was recovered by direct stripping of water and drying to a waxy solid.Preparation of Ag+ Salt of SEP-RM (Ag-SEP-RM) Rhamnolipid

[0109] To 1000 milliliters round bottom flask equipped with a mechanical stirrer, thermocouple, nitrogen inlet / outlet Schlenk line and glass stopper were added 100 grams of SEP-RM (or Na-SEP-RM) rhamnolipid. The solution was allowed to mix at room temperature for 5 minutes, then 400 grams of 0.1 N AgNOs solution was slowly added and the reaction instantaneously changed color with some visible residue forming on the sides of the glass. The reaction was allowed to mix at room temperature for 24 hours. Once the ion exchange reaction was completed, the crude product was transferred to multiple 50 ml_ centrifuge tubes and centrifuged to afford two distinctive layers: 1 ) top aqueous layer and 2) bottom organic layer containing silver rhamnolipid salts. The top layer was extracted and discarded, while the bottom layer was washed with DI H2O (3x). After water washing, all the organic layers were combined and transferred to a recrystallization disc and dried down at 105°C for couple hours. After drying, the final rhamnolipid silver salt product was collected as a brown solid.Additional Preparation and Synthesis of Rhamnolipid SaltsSilver (Ag+) Rhamnolipid Salt

[0110] To 500 milliliters round bottom flask equipped with a large magnetic stir bar was added 37 grams of acidulated SEP-RM (A-SEP-RM) rhamnolipid and 160 grams of dichloromethane. The solution was allowed to mix at room temperature for roughly 45 minutes, until the RL was completely solubilized in the organic solvent. Once the reaction was homogeneous, 5.5 grams of silver (I) oxide was transferred to the flask and the entirereaction was mixed at room temperature for 24 hours. Once the reaction was completed, the crude reaction mixture was slowly pipetted into a stirring 1 .5 liters reservoir of diethyl ether in a 2 liters Erlenmeyer flask to precipitate out the silver rhamnolipid salt. The precipitate was collected using a Buchner funnel with filter paper, then dried down on the rotovap and placed on the high vacuum overnight to remove the traces of the solvent. The final rhamnolipid silver salt produced was collected as a brown solid.Potassium (K+) Rhamnolipid Salt

[0111] To a 4-ounce jar equipped with a large magnetic stir bar was added 11 grams of acidulated SEP-RM (A-SEP-RM) rhamnolipid and 50 grams of deionized water. The stirring was initiated, and the reaction was heterogeneous. At this point, 0.78 grams of potassium hydroxide was added, and the mixture was stirred for 24 hours at room temperature. After 24 hours, the reaction was homogeneous, and the neutralization was completed to generate potassium rhamnolipid salt as an amber liquid.Choline Rhamnolipid Salt

[0112] To a 4-ounce jar equipped with a large magnetic stir bar was added 15 grams of acidulated SEP-RM (A-SEP-RM) rhamnolipid and 60 grams of deionized water. The stirring was initiated, and the reaction was heterogeneous. At this point, 7 grams of choline hydroxide (46 wt.% in water) was added, and the mixture was stirred for 24 hours at room temperature. After 24 hours, the reaction was homogeneous, and the neutralization was completed to generate choline rhamnolipid salt as an amber liquid.Zinc (Zn2+) Rhamnolipid Salt

[0113] To a 4-ounce jar equipped with a large magnetic stir bar was added 79 grams of SEP-RM (or Na-SEP-RM) rhamnolipid and 1 1 grams of zinc (II) chloride (25 wt.% in water). The stirring was initiated, and 100 grams of water was added. The zinc rhamnolipid salt precipitated instantaneously and the reaction was left to mix for 24 hours at room temperature. After the reaction was completed, the crude was filtered using Buchner funnel equipped with a filter paper under vacuum and the collected solid was washed 3 times with deionized water. The product was then transferred to a separate 250 mL round bottom flask and placed on rotary evaporator to remove the traces of water.After stripping off water, the product was placed in a high vacuum overnight. The final zinc rhamnolipid salt was collected as a brown solid.Copper (Cu2+) Rhamnolipid Salt

[0114] To a 4-ounce jar equipped with a large magnetic stir bar was added 80 grams of SEP-RM (or Na-SEP-RM) rhamnolipid and 12 grams of copper (II) chloride (25 wt.% in water). The stirring was initiated, and 100 grams of water was added. The copper rhamnolipid salt precipitated instantaneously and the reaction was left to mix for 24 hours at room temperature. After the reaction was completed, the crude was filtered using Buchner funnel equipped with a filter paper under vacuum and the collected solid was washed 3 times with deionized water. The product was then transferred to a separate 250 mL round bottom flask and placed on rotary evaporator to remove the traces of water. After stripping off water, the product was placed in a high vacuum overnight. The final copper rhamnolipid salt was collected as a blue / green solid.Manganese (Mn2+) Rhamnolipid Salt

[0115] To a 4-ounce jar equipped with a large magnetic stir bar was added 85 grams of SEP-RM (or Na-SEP-RM) rhamnolipid and 15 grams of manganese (II) chloride (25 wt.% in water). The stirring was initiated, and 100 grams of water was added. The manganese rhamnolipid salt precipitated instantaneously and the reaction was left to mix for 24 hours at room temperature. After the reaction was completed, the crude was filtered using Buchner funnel equipped with a filter paper under vacuum and the collected solid was washed 3 times with deionized water. The product was then transferred to a separate 250 mL round bottom flask and placed on rotary evaporator to remove the traces of water. After stripping off water, the product was placed in a high vacuum overnight. The final manganese rhamnolipid salt was collected as a brown solid.Calcium (Ca2+) Rhamnolipid Salt

[0116] To a 2-liter round bottom flask equipped with a large magnetic stir bar was added 574 grams of SEP-RM (or Na-SEP-RM) rhamnolipid and 55 grams of calcium (II) chloride (25 wt.% in water). The stirring was initiated, and 550 grams of water was added. The calcium rhamnolipid salt precipitated instantaneously and the reaction was left to mixfor 24 hours at room temperature. After the reaction was completed, the remaining aqueous (top) layer was decanted and the rhamnolipid (bottom) layer was washed 3 times with deionized water, followed by decantation of the aqueous layer. The remaining rhamnolipid layer was transferred to centrifuge tubes to be spun down and the supernatant was further decantated. The rhamnolipid layer was then transferred to a 1 - liter round bottom flask and placed on rotary evaporator to remove the residual water. After stripping off water, the product was placed in a high vacuum overnight. The final calcium rhamnolipid salt was collected as a brown solid.Magnesium (Mg2+) Rhamnolipid Salt

[0117] To a 1 -liter round bottom flask equipped with a large magnetic stir bar was added 1 2 grams of SEP-RM (or Na-SEP-RM) rhamnolipid and 10 grams of magnesium (II) chloride (25 wt.% in water). The stirring was initiated, and 100 grams of water was added. The magnesium rhamnolipid salt did not precipitate and instead was soluble in water. Additional water was added to the reaction to see if it would precipitate out the salt but was not successful. The reaction was mixed for 24 hours at room temperature. After the reaction was completed, the water was removed using rotary evaporator and then the product was placed in a high vacuum overnight. The final magnesium rhamnolipid salt was collected as waxy solid.EXAMPLE 1 : Curcumin-Loaded Rhamnosomes (Mono-Rhamnolipid versus Di- Rhamnolipid)

[0118] A study was conducted to assess the production of rhamnolipid containing liposomes (i.e., rhamnosomes), using column chromatography purified monorhamnolipids and di-rhamnolipids. The column chromatography method comprised of dissolving acid version of rhamnolipid in CHCh and liquid injecting it into automated column chromatography system (CombiFlash® NextGen 300+) for a separation using silica gel stationary phase. The mobile phase used was gradient 0 to 50% MeOH:CHCl3 (v / v). The fractions collected were visualized using thin-layer chromatography (TLC) on silica gel places with a mobile phase consisting of CHCl3:MeOH:H2O (65:15:2, v / v / v) and iodine chamber. After examining the fractions from column chromatography using TLC,the desired pure mono-RL and pure di-RL fractions were collected in round bottom flasks, and the solvent was stripped off using rotary evaporation to afford dry final products. The rhamnosomes were prepared using a thin-film hydration (TFH) method, and passively loaded with curcumin (i.e., curcumin was encapsulated during rhamnosome formation). In this example, the TFH method comprised casting a dried film from a solution comprising a mixture of rhamnolipid and curcumin at a ratio of about 87 / 13 from solvent (2:1 v / v chloroform / methanol) onto a round bottom flask via rotary evaporation, and then hydrating the film with unbuffered H2O at ambient temperature via rotation of the flask in the presence of 2 mm glass beads for 20 minutes. The concentration of rhamnolipid in the hydrated solution was about 1 .5 mg / mL. A portion of the hydrated solution was subjected to filtration through a 0.45 pm syringe filter.

[0119] The resultant rhamnosomes, loaded with curcumin, were subject to Dynamic Light Scattering (DLS) analysis, wherein mean particle diameter and PDI were determined for compositions diluted by a factor 10x with deionized water. The results are as follows: a Mean Particle Diameter of 386 nm, and a PDI of 0.25, for curcumin-loaded rhamnosomes was produced using mono-rhamnolipid (characterized day of fabrication (initial)); a Mean Particle Diameter of 148 nm, and a PDI of 0.14, for curcumin-loaded rhamnosomes, was produced using mono-rhamnolipid (filtered, measured after 4 days at ambient temperature); a Mean Particle Diameter of 453 nm, and a PDI of 0.28, for curcumin-loaded rhamnosomes, was produced using di-rhamnolipid (characterized day of fabrication (initial)); and a Mean Particle Diameter of >10K nm, and a PDI of 0.74, for curcumin-loaded rhamnosomes, was produced using di-rhamnolipid (filtered after 4 days). Figure 1 provides corresponding curcumin retention (%) values for curcumin- loaded rhamnosomes produced under this example. Curcumin retention was measured by sampling the supernatant of statically held samples, diluting with ethanol, measuring absorbance at 425 nm, and calculating curcumin concentration using a calibration curve generated from serial dilutions of curcumin in ethanol of known concentration.EXAMPLE 2: Spontaneity for Narrow PDI I Small Particles Using the Thin Film Hydration Method

[0120] A study was conducted to compare the production of rhamnosomes and liposomes (without active (i.e. unloaded)) fabricated using, in this example, the thin-film hydration method. Liposomes were produced using either Soy Phosphatidylcholine (Soy PC) or Dipalmitoyl Phosphatidylcholine (DPPC). Rhamnosomes were produced using either acidulated SEP-RM (A-SEP-RM), purified mono-RL, purified di-RL, or a 1 :1 m / m ratio of purified mono-RL and purified di-RL. All thin film hydrations were conducted with unbuffered H2O at ambient temperature except for DPPC, which was conducted at elevated temperatures due to this lipid’s elevated phase transition (gel-to-liquid) temperature of about 42 °C.

[0121] The resultant liposomes and rhamnosomes were subject to Dynamic Light Scattering analysis (DLS), wherein mean particle diameter and PDI were determined. The results are summarized in Table 1 below.Table 1aN.V.: Not Viable analysis based on poor DLS data quality (related to poor spontaneity)EXAMPLE 3: Spontaneity in Fabrication of Liposomes and Rhamnosomes using Ethanol Injection Method

[0122] A study was conducted to assess spontaneity in the fabrication of liposomes and rhamnosomes using the ethanol injection (El) method. The El method comprises the controlled injection of a dilute solution of lipid (and optionally an active) in ethanol into an aqueous liquid such as unbuffered water, saline, or buffer, with continuous stirring. The concentration of lipid in ethanol can range from very dilute to highly concentrated. For the example here, the lipid concentration was about 33 mg / mL, the ratio of ethanol solution to final volume upon injection was 1 :20, unbuffered water was used, and the time to complete the injection was about 40 seconds. As indicated below in Table 2, the following lipids were used for the production of liposomes: purified soy phosphatidylcholine (Soy PC); and Dipalmitoyl Phosphatidylcholine (DPPC). Likewise, the following rhamnolipids were used in the production of rhamnosomes: BWAR rhamnolipid; acidulated SEP-RM (A-SEP-RM); purified mono-rhamnolipid; purified dirhamnolipid; and 1 :1 purified mono - 1 di-rhamnolipid. BWAR is a rhamnolipid composition pulled at an intermediate level of purification during the multi-step preparation of SEP- RM, is in an acidulated (non-salt) form, and comprises about 40-60% water.

[0123] The resultant liposomes and rhamnosomes were subject to Dynamic Light Scattering analysis, wherein mean particle diameter and PDI were determined. The results are summarized below in Table 2:Table 2EXAMPLE 4: Spontaneity in Fabrication of Rhamnosomes using Ethanol Injection Method in Combination with Phosphate-buffered saline (PBS) Buffer System (Acidulated SEP-RM (A-SEP-RM) Rhamnolipid)

[0124] A study was conducted to assess spontaneity in the fabrication of rhamnosomes using the ethanol injection method in combination with a buffer system. As indicated below in Table 3, the lipid was acidulated SEP-RM (A-SEP-RM) Rhamnolipid, and the aqueous conditions tested included phosphate-buffered saline (PBS) at different pHs, as well as no buffer in the presence of 150 and 15 mM aqueous NaCL

[0125] The resultant rhamnosomes were subject to Dynamic Light Scattering analysis, wherein mean particle diameter and PDI were determined. The results are summarized below in Table 3:Table 3EXAMPLE 5: Spontaneity in Fabrication of Rhamnosomes using Ethanol Injection Method in Combination with Phosphate-buffered saline (PBS) Buffer System (Purified Mono- and Di- Rhamnolipids)

[0126] A study was conducted to assess spontaneity in the fabrication of rhamnosomes using the ethanol injection method in combination with a buffer system. As indicated below in Table 4, the lipids used included purified mono-rhamnolipid, purified di-rhamnolipid, and 1 :1 purified mono- / di-rhamnolipid. The aqueous conditions tested included Phosphate-buffered saline (PBS) at different pHs, as well as no buffer in the presence of 15 mM NaCI.

[0127] The resultant rhamnosomes were subject to Dynamic Light Scattering analysis, wherein mean particle diameter and PDI were determined. The results are summarized below in Table 4:Table 4EXAMPLE 6: Fabrication of Rhamnosomes using Ethanol Injection Method in Combination with Phosphate Buffer

[0128] A study was conducted to assess spontaneity in the fabrication of rhamnosomes using the ethanol injection method in combination with 0.01 M Phosphate Buffer (PB) at different pHs. The lipids used included: acidulated SEP-RM (A-SEP-RM); purified mono-rhamnolipid; and purified di-rhamnolipid.

[0129] The resultant rhamnosomes were subject to Dynamic Light Scattering analysis, wherein the mean particle diameter was determined. The results are as follows: Figure 2 shows the resultant Mean Particle Diameter (nm) for acidulated SEP-RM (A- SEP-RM) in 0.01 M Phosphate Buffer (PB) at different pHs (the day of formation, and 1 week after formation); Figure 3 shows the resultant Mean Particle Diameter (nm) for purified mono-rhamnolipid in 0.01 M Phosphate Buffer (PB) at different pHs (the day of formation, and 1 week after formation); and Figure 4 shows the resultant Mean Particle Diameter (nm) for purified di-rhamnolipid in 0.01 M Phosphate Buffer (PB) at different pHs (the day of formation, and 1 week after formation).EXAMPLE 7: Fabrication of Rhamnosomes using Ethanol Injection Method in Combination with Phosphate Buffer and Na-Rhamnolipid (Na-SEP-RM)

[0130] A study was conducted to assess spontaneity in the fabrication of rhamnosomes using the ethanol or H2O injection method in combination with 0.01 M Phosphate Buffer (PB) at different pHs. The lipid used was Na-SEP-RM. The resultant rhamnosomes were subject to Dynamic Light Scattering analysis, wherein the mean particle diameter was determined. The results are set forth in Figures 5-6, wherein: Figure 5 shows the resultant Mean Particle Diameter (nm) for Na-SEP-RM (from Ethanol) in 0.01 M Phosphate Buffer (PB) at different pHs (the day of formation, and 1 week after formation); and Figure 6 shows the resultant Mean Particle Diameter (nm) for Na-SEP- RM (from H2O) in 0.01 M Phosphate Buffer (PB) at different pHs (the day of formation, and 1 week after formation).EXAMPLE 8: Fabrication of Rhamnolipid Modified Soy Phosphatidylcholine (PC) Liposomes

[0131] A study was conducted to assess the fabrication of rhamnolipid modified Soy Phosphatidylcholine (Soy PC) liposomes, prepared using the ethanol injection method (at various pH values) and acidulated SEP-RM (A-SEP-RM) rhamnolipid. The rhamnolipid:Soy PC ratios tested included 1 :0, 3:1 , 1 :1 , 1 :3, 1 :9, 1 :19, and 0:1. The resultant rhamnolipid modified liposomes were subject to Dynamic Light Scattering analysis, wherein mean particle diameter and PDI were determined. The results are set forth in Figures 7 - 20.

[0132] Figures 7 and 8 respectively show the Mean Particle Diameter and PDI for rhamnosomes prepared using the ethanol injection method (unbuffered, 150mM aqueous NaCI, or 0.01 M phosphate buffer at various pH values) and acidulated SEP-RM (A-SEP- RM) rhamnolipid (measured the day of formation, and 1 week after formation).

[0133] Figures 9 and 10 respectively show the Mean Particle Diameter and PDI for liposomes prepared using the ethanol injection method (unbuffered, 150mM aqueous NaCI, or 0.01 M phosphate buffer at various pH values) and Soy PC (measured the day of formation, and 1 week after formation).

[0134] Figures 1 1 and 12 respectively show the Mean Particle Diameter and PDI for acidulated SEP-RM (A-SEP-RM) rhamnolipid (RL) modified liposomes prepared using the ethanol injection method (unbuffered, 150mM aqueous NaCI, or 0.01 M phosphate buffer at various pH values)and 3:1 RL / Soy PC (measured the day of formation, and 1 week after formation).

[0135] Figures 13 and 14 respectively show the Mean Particle Diameter and PDI for acidulated SEP-RM (A-SEP-RM) rhamnolipid (RL) modified liposomes prepared using the ethanol injection method (unbuffered, 150mM aqueous NaCI, or 0.01 M phosphate buffer at various pH values)and 1 :1 RL / Soy PC (measured the day of formation, and 1 week after formation).

[0136] Figures 15 and 16 respectively show the Mean Particle Diameter and PDI for acidulated SEP-RM (A-SEP-RM) rhamnolipid (RL) modified liposomes prepared using the ethanol injection method (unbuffered, 150mM aqueous NaCI, or 0.01 M phosphate buffer at various pH values)and 1 :3 RL / Soy PC (measured the day of formation, and 1 week after formation).

[0137] Figures 17 and 18 respectively show the Mean Particle Diameter and PDI for acidulated SEP-RM (A-SEP-RM) rhamnolipid (RL) modified liposomes prepared using the ethanol injection method (unbuffered, 150mM aqueous NaCI, or 0.01 M phosphate buffer at various pH values)and 1 :9 RL / Soy PC (measured the day of formation, and 1 week after formation).

[0138] Figures 19 and 20 respectively show the Mean Particle Diameter and PDI for acidulated SEP-RM (A-SEP-RM) rhamnolipid (RL) modified liposomes prepared using the ethanol injection method (unbuffered, 150mM aqueous NaCI, or 0.01 M phosphate buffer at various pH values)and 1 :19 RL / Soy PC (measured the day of formation, and 1 week after formation).EXAMPLE 9: Effect of Cholesterol on Rhamnosome Fabrication

[0139] A study was conducted to assess the effect of cholesterol on the fabrication of rhamnosomes, prepared using the ethanol injection method and acidulated SEP-RM (A-SEP-RM) rhamnolipid (RL) in the presence of increasing concentrations of cholesterol(relative to total lipid + sterol). The resultant rhamnolipid modified liposomes were subject to Dynamic Light Scattering analysis, wherein mean particle diameter and PDI were determined. The results are set forth in Figures 21 and 22.

[0140] Figure 21 shows the Mean Particle Diameter for rhamnosomes prepared using the ethanol injection method (unbuffered H2O) and acidulated SEP-RM (A-SEP- RM) rhamnolipid together with various amounts of cholesterol (measured the day of formation, and 1 week after formation).

[0141] Figure 22 shows the PDI for rhamnosomes prepared using the ethanol injection method (unbuffered H2O) and acidulated SEP-RM (A-SEP-RM) rhamnolipid together with various amounts of cholesterol (measured the day of formation, and 1 week after formation).EXAMPLE 10: Effect of Cholesterol on Rhamnosome Fabrication

[0142] A study was conducted assess the effect of aqueous conditions on the fabrication of cholesterol-comprising rhamnosomes. The rhamnosomes were prepared using the ethanol injection method ( unbuffered water, 150mM aqueous NaCI, 0.01 M phosphate buffer at various pH values) and acidulated SEP-RM (A-SEP-RM) rhamnolipid (RL) in the presence of cholesterol (80:20 RL / cholesterol). The resultant rhamnolipid modified liposomes were subject to Dynamic Light Scattering analysis, wherein mean particle diameter and PDI were determined. The results are set forth in Figures 23 and 24.

[0143] Figure 23 shows the Mean Particle Diameter for rhamnosomes prepared using the ethanol injection method (unbuffered, 150mM aqueous NaCI, or 0.01 M phosphate buffer at various pH values) and acidulated SEP-RM (A-SEP-RM) rhamnolipid (RL) in the presence of cholesterol (80:20 RL / cholesterol).

[0144] Figure 24 shows the PDI for rhamnosomes prepared using the ethanol injection method (unbuffered, 150mM aqueous NaCI, or 0.01 M phosphate buffer at various pH values) and acidulated SEP-RM (A-SEP-RM) rhamnolipid (RL) in the presence of cholesterol (80:20 RL / cholesterol).EXAMPLE 11 : Impact of Active Loading on Rhamnosome Fabrication

[0145] A study was conducted to assess the impact of active loading on rhamnosome formation. In this study, Curcumin was used as representative active. Rhamnosomes were prepared using the ethanol injection method and acidulated SEP- RM (A-SEP-RM) rhamnolipid (RL). Curcumin was present during formation of the rhamnosomes (passive loading) at 0%, 5%, and 15% by weight on rhamnolipid. The resultant rhamnosomes were subject to Dynamic Light Scattering analysis, wherein mean particle diameter and PDI were determined. The results are set forth in Figures 25-27.

[0146] Figure 25 shows the Mean Particle Diameter for rhamnosomes prepared (unbuffered, 150mM aqueous NaCI, or 0.01 M phosphate buffer at various pH values) in the presence of 0 wt% Curcumin (measured the day of formation, and 1 week after formation (stored at room temperature)).

[0147] Figure 26 shows the Mean Particle Diameter for rhamnosomes prepared (unbuffered, 150mM aqueous NaCI, or 0.01 M phosphate buffer at various pH values) in the presence of 5 wt% Curcumin (measured the day of formation, and 1 week after formation (stored at room temperature, and 10°C)).

[0148] Figure 27 shows the Mean Particle Diameter for rhamnosomes prepared (unbuffered, 150mM aqueous NaCI, or 0.01 M phosphate buffer at various pH values) in the presence of 15 wt% Curcumin (measured the day of formation, and 1 week after formation (stored at room temperature, and 10°C)).EXAMPLE 12: Fabrication of Mixed Di-RL Soy PC Liposomes

[0149] A study was conducted to assess the fabrication of purified di-rhamnolipid modified Soy Phosphatidylcholine (Soy PC) liposomes, prepared using the ethanol injection method (at various pH values). The purified di-rhamnolipid:Soy PC ratio tested was 1 :3. The resultant rhamnolipid modified liposomes were subject to Dynamic Light Scattering analysis, wherein mean particle diameter and PDI were determined. The results are set forth in Figures 28-35.

[0150] Figures 28 and 29 respectively show the Mean Particle Diameter and PDI for purified di-rhamnolipid (Di-RL) rhamnosomes prepared using the ethanol injectionmethod (unbuffered, 150mM aqueous NaCI, or 0.01 M phosphate buffer at various pH values) and Di-RL (measured the day of formation, and 1 week after formation).

[0151] Figures 30 and 31 respectively show the Mean Particle Diameter and PDI for Soy PC liposomes prepared using the ethanol injection method (unbuffered, 150mM aqueous NaCI, or 0.01 M phosphate buffer at various pH values) and Soy PC (measured the day of formation, and 1 week after formation).

[0152] Figures 32 and 33 respectively show the Mean Particle Diameter and PDI for purified di-rhamnolipid (Di-RL) modified Soy PC liposomes prepared using the ethanol injection method (unbuffered, 150mM aqueous NaCI, or 0.01 M phosphate buffer at various pH values) and a 1 :3 Di-RL / Soy PC ratio (measured the day of formation, and 1 week after formation).

[0153] Figures 34 and 35 compares acidulated SEP-RM (A-SEP-RM) rhamnolipid modified Soy PC liposomes prepared using the ethanol injection method (unbuffered, 150mM aqueous NaCI, or 0.01 M phosphate buffer at various pH values) and a 1 :3 acidulated SEP-RM (A-SEP-RM) RL / Soy PC ratio, versus purified di-rhamnolipid (Di-RL) modified Soy PC liposomes prepared using the ethanol injection method (unbuffered in the presence of 150mM NaCI, and at various pH values) and a 1 :3 Di-RL / Soy PC ratio.

[0154] Specifically, Figures 34 A-B compare the Mean Particle Diameter of acidulated SEP-RM (A-SEP-RM) rhamnolipid modified Soy PC liposomes prepared using the ethanol injection method (unbuffered, 150mM aqueous NaCI, or 0.01 M phosphate buffer at various pH values) and a 1 :3 acidulated SEP-RM (A-SEP-RM) rhamnolipid RL / Soy PC ratio, versus purified di-rhamnolipid (Di-RL) modified Soy PC liposomes prepared using the ethanol injection method (unbuffered, 150mM aqueous NaCI, or 0.01 M phosphate buffer at various pH values) and a 1 :3 Di-RL / Soy PC ratio (measured the day of formation, and 1 week after formation).

[0155] Figures 35 A-B compares the PDI of acidulated SEP-RM (A-SEP-RM) rhamnolipid modified Soy PC liposomes prepared using the ethanol injection method (unbuffered, 150mM aqueous NaCI, or 0.01 M phosphate buffer at various pH values) and a 1 :3 acidulated SEP-RM (A-SEP-RM) RL / Soy PC ratio, versus purified di-rhamnolipid (Di-RL) modified Soy PC liposomes prepared using the ethanol injection method(unbuffered, 150mM aqueous NaCI, or 0.01 M phosphate buffer at various pH values) and a 1 :3 Di-RL / Soy PC ratio (measured the day of formation, and 1 week after formation).EXAMPLE 13: Mixed di-rhamnolipid / Soy PC liposomes (Phosphate Buffer)

[0156] A study was conducted to assess the fabrication of mixed purified dirhamnolipid / Soy PC liposomes in the presence 0.01 M Phosphate Buffer (pH 5.0) using the ethanol injection method. Figures 36 A-B show the Mean Particle Diameter and PDI of the resultant liposomes (measured the day of formation, and 1 week after formation).EXAMPLE 14: Effect of Cholesterol on Rhamnosome Formation Using Purified Di- Rhamnolipid

[0157] A study was conducted to assess the effect of cholesterol on the formation of rhamnosomes prepared using the ethanol injection method and purified di-rhamnolipid (unbuffered, 150mM aqueous NaCI, or 0.01 M phosphate buffer at various pH values). The di-rhamnolipid / cholesterol ratio tested was 80:20. Figures 37 A-B show the Mean Particle Diameter (Figure 37 A) and PDI (Figure 37 B) of the resultant liposomes (measured the day of formation, and 1 week after formation).EXAMPLE 15: Comparison of Stability of Curcumin-Loaded Rhamnosomes to Phospholipid-Derived Curcumin-Loaded Liposomes

[0158] A comparative study was conducted to assess the stability of curcumin- loaded rhamnosomes versus curcumin-loaded liposomes. Rhamnosomes were fabricated using the ethanol injection method, wherein 0.50 mL of a solution comprising acidulated SEP-RM (A-SEP-RM) rhamnolipid and curcumin in ethanol was injected with stirring into 9.50 mL of unbuffered water or aqueous phosphate buffer at ambient temperature to achieve passive loading of active. The injection rate was about 0.7 mL / minute and was conducted at ambient temperature. In the same way, curcumin- loaded liposomes were fabricated using either soy phosphatidylcholine or DPPC instead of rhamnolipid. For DPPC, ethanol solution injection was conducted with water or buffer pre-heated above 45QC. For soy phosphatidylcholine, unbuffered water was adjusted with 0.05 mL of 0.01 M aqueous H3PO4 so as to achieve comparable pH in produced liposome compositions to rhamnosome compositions prepared from acidulated SEP-RM(A-SEP-RM). For DPPC, unbuffered water was adjusted with 0.1 ml_ of 0.01 M aqueous H3PO4 so as to achieve comparable pH in produced liposome compositions to rhamnosome compositions prepared from acidulated SEP-RM (A-SEP-RM). In these fabrications, the concentration of rhamnolipid or phospholipid in ethanol prior to injection was in the range of about 32-35 mg / mL. Samples of the resultant compositions were then stored at ambient temperature and at ~10SC and analyzed over time for particle size, PDI, and amount of curcumin remaining encapsulated. Encapsulated curcumin was measured by sampling the supernatant of sample after centrifugation at 5000 rpm for 3 minutes, diluting in ethanol, and quantifying non-precipitated curcumin by spectrophotometric analysis at 425 nm, using a calibration curve generated from serial dilutions of curcumin in ethanol of known concentration.

[0159] Figures 38 A-B shows the initial mean particle diameter (Figure 38 A) and PDI (Figure 38 B) of rhamnosomes fabricated without active and with increasing loading levels of curcumin (fabricated from unbuffered H2O or 0.01 M phosphate buffer at various pH values).

[0160] Figures 39 A-B shows the change in mean particle diameter (Figure A) and PDI (Figure B) of rhamnosomes fabricated with 5 wt% curcumin on lipid (fabricated from unbuffered H2O or 0.01 M phosphate buffer at various pH values) with time upon storage at ambient temperature (RT) or 10QC.

[0161] Figure 40 shows the change in encapsulated curcumin for rhamnosomes fabricated with 5 wt% curcumin on lipid (fabricated from unbuffered H2O or 0.01 M phosphate buffer at various pH values) with time upon storage at ambient temperature (RT) or 10QC.

[0162] Figures 41 A-B show the change in mean particle diameter (Figure 41 A) and PDI (Figure 41 B) of rhamnosomes fabricated with 10 wt% curcumin on lipid (fabricated from unbuffered H2O or 0.01 M phosphate buffer at various pH values) with time upon storage at ambient temperature (RT) or 10QC.

[0163] Figure 42 shows the change in encapsulated curcumin for rhamnosomes fabricated with 10 wt% curcumin on lipid (fabricated from unbuffered H2O or 0.01 Mphosphate buffer at various pH values) with time upon storage at ambient temperature (RT) or 10QC.

[0164] Figures 43 A-B shows the change in encapsulated curcumin for rhamnosomes fabricated with increasing loading levels of curcumin on lipid (fabricated from unbuffered H2O (Figure 43 A) or 0.01 M phosphate buffer at pH 6.0 (Figure 43 B)) with time upon storage at ambient temperature (RT) or 10SC.

[0165] Figures 44 A-B shows the change in encapsulated curcumin for soy phosphatidylcholine liposomes fabricated with increasing loading levels of curcumin on lipid (fabricated from HsPC -adjusted H2O (Figure 44A) or 0.01 M phosphate buffer at pH 6.0 (Figure 44 B)) with time upon storage at ambient temperature (RT) or 10QC. Comparison of these data to those in Figures 43A-B indicates that curcumin-loaded liposomes fabricated from soy phosphatidylcholine are significantly less stable than curcumin-loaded rhamnosomes, fabricated from acidulated SEP-RM (A-SEP-RM), in terms of retention of encapsulated active.

[0166] Figures 45 A-B shows the change in encapsulated curcumin for DPPC liposomes fabricated with increasing loading levels of curcumin on lipid (fabricated from HsPC -adjusted H2O (Figure 45 A) or 0.01 M phosphate buffer at pH 6.0 (Figure 45 B)) with time upon storage at ambient temperature (RT) or 10QC. Comparison of these data to those in Figures 43 A-B indicates that curcumin-loaded liposomes fabricated from DPPC are significantly less stable than curcumin-loaded rhamnosomes, fabricated from acidulated SEP-RM (A-SEP-RM), in terms of retention of encapsulated active.EXAMPLE 16: Stability of Curcumin-Loaded Rhamnosomes Derived from Na SEP- RM Rhamnolipid, Purified Mono-RL or Purified Di-RL

[0167] A comparative study was conducted to assess the stability of curcumin- loaded rhamnosomes fabricated from Na-SEP-RM rhamnolipid (RL), purified mono-RL, or purified di-RL using the ethanol injection method. The conditions used to fabricate and characterize rhamnosomes was the same as described in Example 15, except for buffer conditions, which were chosen to produce fabricated rhamnosome compositions with pH values comparable to those achieved with acidulated SEP-RM (A-SEP-RM) fabricated from pH 6.0 buffer. For Na-SEP-RM rhamnolipid (RL), 0.01 M phosphate buffer pH 3.0-M- was used. For purified mono-RL and purified di-RL, 0.01 M phosphate buffer pH 5.5 was used.

[0168] Figure 46 shows the change in encapsulated curcumin for rhamnosomes prepared from Na-SEP-RM rhamnolipid (RL) and pH 3.0 buffer, fabricated with increasing loading levels of curcumin on lipid, with time upon storage at ambient temperature (RT) or 10SC.

[0169] Figure 47 shows the change in encapsulated curcumin for rhamnosomes prepared from purified mono-RL and pH 5.5 buffer, fabricated with increasing loading levels of curcumin on lipid, with time upon storage at ambient temperature (RT) or 10QC.

[0170] Figure 48 shows the change in encapsulated curcumin for rhamnosomes prepared from purified di-RL and pH 5.5 buffer, fabricated with increasing loading levels of curcumin on lipid, with time upon storage at ambient temperature (RT) or 10QC.EXAMPLE 17: Control of Curcumin-Loaded Rhamnosome Particle Size and Stability by Means of Post-Fabrication pH Adjustment

[0171] A study was conducted to demonstrate the stabilization of particle size and curcumin encapsulation of rhamnosomes fabricated from acidulated SEP-RM (A-SEP- RM), wherein the rhamnosomes were fabricated at an initial pH to produce small particles of less than 100 nm, followed by post-fabrication adjustment to lower pH to achieve stabilization. Fabrication was conducted via the ethanol injection method, wherein 1 .00 mL of a solution comprising acidulated SEP-RM (A-SEP-RM) rhamnolipid and curcumin (5 wt% on lipid) in ethanol was injected with stirring into 9.00 mL of 0.01 M phosphate buffer pH 6.5 at ambient temperature to achieve passive loading of active. The injection rate was about 0.7 mL / minute and was conducted at ambient temperature. The resulting composition, with a pH of 6.25, was characterized by dynamic light scattering and curcumin assay. Then 5.0 mL of this aqueous composition was adjusted to pH 5.2 by addition of 8.0 mL of 0.01 M phosphate buffer pH 3.0 with stirring at a rate of about 1.5 mL per minute. The resulting adjusted composition was then characterized by dynamic light scattering and curcumin assay. Both compositions (i.e., before and after pH adjustment) were then monitored for changes with time upon storage at ambient temperature (RT) or 10°C, wherein curcumin was assayed in terms of remaining curcuminin supernatant (non-precipitated) upon centrifugation at 5000 rpm for 3 minutes. Results of the study are summarized in the table below. Particle size is retained over time for the post-adjusted composition. Relative to pre-adjusted composition, the post-adjusted composition displayed a large improvement in % curcumin retention.Table 5.EXAMPLE 18: Control of Rhamnosome and Rhamnolipid-Modified Liposome Particle Size and Stability by Means of Post-Fabrication pH Adjustment

[0172] Aqueous compositions comprising rhamnosomes or rhamnolipid-modified liposomes with various ratios of rhamnolipid to phospholipid are prepared by means of ethanol injection method, aqueous injection method, thin-film hydration method, or other methods of fabrication used by those skilled in the art. In some instances, the compositions further comprise an active. Fabrication is conducted at a first pH that affordsa relatively smaller particle size compared to that achieved by comparable conditions but at lower pH. The resulting aqueous composition is then adjusted to lower pH by means of the addition of aqueous acid or buffer. The resulting aqueous composition displays improved stability in terms of particle size, PDI, and / or actives retention compared to a composition that has not been pH adjusted. The composition thereby possesses both relatively smaller particle size in combination with good stability.EXAMPLE 19: Alteration of Actives Delivery to Substrate by Means of Controlling Rhamnosome or Rhamnolipid-Modified Liposome Particle Characteristics Through Post-Fabrication pH Adjustment

[0173] An aqueous composition produced by the method described in Example 18, comprising at least one active, is prepared. The composition is applied to a substrate such as human or animal skin or a plant leaf, or synthetic membrane, by means of a Franz cell diffusion apparatus. The sample is maintained in contact with the substrate for 24 hrs. The substrate is removed from the Franz cell and rinsed with water. The substrate is then characterized in terms of total deposition of actives as well as profile of active deposition across a cross-section of the substrate. The composition is found to show enhanced ability to penetrate and deliver active into deeper layers or distances within the substrate compared to a composition prepared by a conventional fabrication without pH adjustment that produces larger particle size.Example 20: Encapsulation of Water-Soluble Actives in Rhamnosome or Rhamnolipid-Modified Liposome by Means of Aqueous Injection Method

[0174] Aqueous compositions comprising water-soluble actives loaded rhamnosomes or rhamnolipid-modified liposomes free of phospholipids or with various ratios of rhamnolipid to phospholipid are prepared by means of an aqueous injection method, wherein a solution of lipid and active in water at a relatively higher pH is injected into an aqueous condition of relatively lower pH. The pH of the solution of lipid in water is at or above a pH that enables full solubility of the lipid or lipid combination and active. The fabrication method enables a high degree of efficiency in encapsulation of water soluble active and does not require the use of organic solvents.Example 21 : Fabrication of Mixed Rhamnolipid / Phospholipid Liposomes and / or Nanoparticles by Means of Aqueous Injection Method

[0175] A study was conducted to demonstrate the fabrication of liposomes or nanoparticles comprising mixtures of rhamnolipid and phospholipids by means of an aqueous injection method, wherein water-insoluble phospholipids were solubilized in aqueous rhamnolipid solutions and then injected with continuous stirring into a lower pH aqueous buffer at ambient temperature to form liposomes. Aqueous solutions of rhamnolipid and phospholipid mixtures were prepared by combining solid phospholipids with an aqueous solution comprising 26.2 wt.% sodium rhamnolipid (assayed by HPLC analysis) and then gently mixing for 48 hours at ambient temperature. For the examples here, when purified soy phosphatidylcholine (soy PC) was used, clear solutions free of haze were obtained. When soy lecithin (90%, powder) was used, a slightly hazy solution resulted and therefore the liquid was subject to centrifugation to clarify the liquid. The aqueous mixtures of rhamnolipids and phospholipids were then diluted in DI H2O and filtered through 0.45 pm cartridge to remove any trace incidental particles prior to use in injection. For the examples here, the total lipid concentration was about 33 mg / mL, the volume injected was 0.5 mL, the volume of the lower pH aqueous buffer was 9.5 mL, and the time to complete the injection was about 40 seconds. The resultant particles were subject to Dynamic Light Scattering analysis, wherein mean particle diameter and PDI were determined. The results are summarized in Table 6 below.Table 6.Example 22: Fabrication of Rhamnosomes and Mixed Rhamnolipid / Phospholipid Liposomes that Further Comprise Cholesterol by Means of Aqueous Injection Method

[0176] A study was conducted to demonstrate that the method described in Example 21 can be utilized to produce rhamnosomes, liposomes, and / or nanoparticles that comprise cholesterol. Aqueous solutions of rhamnolipids and cholesterol, and in some cases, further comprising soy PC, were prepared by combining solid cholesterol with aqueous lipid solutions (26.2% Na rhamnolipid or solutions further comprising soy PC as described in Example 21 ) and heating in a 70 °C oven for several hours, with occasionally mixing, until clear solutions were obtained. The aqueous mixtures were then diluted in DI H2O and filtered through 0.45 pm cartridge to remove any trace incidental particles prior to use in injection. The results are summarized in Table 7 below:Table 7Example 23: Fabrication of Zn2+ Rhamnosomes using Ethanol Injection Method

[0177] A study was conducted to assess the fabrication of Zn2+ rhamnosomes, prepared using the ethanol injection method and Zn2+ salt of SEP-RM (Zn-SEP-RM) rhamnolipid. For this example, the lipid concentration in ethanol was about 40 mg / mL, the ratio of ethanol solution to final volume upon injection was 1 :20, unbuffered water was used, and the time to complete the injection was about 40 seconds. The resultant rhamnosomes were subject to Dynamic Light Scattering analysis. Initial mean particle diameter was 130 nm and PDI was 0.13. After 11 days at ambient temperature, mean particle diameter was 102 nm and PDI was 0.16.Example 24 (Comparative): Stability of Zn2+ Rhamnosomes Fabricated using Ethanol Injection Method, 0.01 M ZnCI Aqueous Solution, and Acidulated Rhamnolipid

[0178] A study was conducted to assess the fabrication stability of Zn2+ rhamnosomes, prepared using prepared using the ethanol injection method and acidulated SEP-RM (A-SEP-RM) rhamnolipid. For the example here, the lipid concentration in ethanol was about 33 mg / mL, the ratio of ethanol solution to final volume upon injection was 1 :20, 0.01 M aqueous ZnCI was used, and the time to complete the injection was about 40 seconds. The resultant rhamnosomes were subject to Dynamic Light Scattering analysis. Initial mean particle diameter was 442 nm and PDI was 0.29. Inspection after 7 days at ambient temperature revealed substantial precipitation. Characterization of this composition by re-suspending, diluting in H2O, and analyzing by dynamic light scattering was not viable.Example 25: Fabrication of Cu2+ Rhamnosomes using Ethanol Injection Method

[0179] A study was conducted to assess the fabrication of Cu2+ rhamnosomes, prepared using the ethanol injection method and Cu2+ salt of SEP-RM (Cu-SEP-RM) rhamnolipid. For the example here, the lipid concentration in ethanol was about 40 mg / mL, the ratio of ethanol solution to final volume upon injection was 1 :20, unbuffered water was used, and the time to complete the injection was about 40 seconds. The resultant rhamnosomes were subject to Dynamic Light Scattering analysis. Initial mean particle diameter was 102 nm and PDI was 0.09. After 8 days at ambient temperature, mean particle diameter was 154 nm and PDI was 0.09.Example 26: Fabrication of Ag+ Rhamnosomes using Ethanol Injection Method

[0180] A study was conducted to assess the fabrication of Ag-i- rhamnosomes, prepared using the ethanol injection method and Ag+ salt of SEP-RM (Ag-SEP-RM) rhamnolipid. For the example here, the preparation of about 40 mg / mL rhamnolipid Ag salt in ethanol resulted in a cloudy solution. Filtration of this solution through a 0.45 urn membrane afforded a clear solution. The ratio of ethanol solution to final volume upon injection was 1 :20, unbuffered water was used, and the time to complete the injection was about 40 seconds. The resultant rhamnosomes were subject to Dynamic Light Scatteringanalysis. Initial mean particle diameter was 82 nm and PDI was 0.14. After 11 days at ambient temperature, mean particle diameter was 66 nm and PDI was 0.33.Example 27: Fabrication of Ca2+ Rhamnosomes using Ethanol Injection Method

[0181] A study was conducted to assess the fabrication of Ca2+ rhamnosomes, prepared using the ethanol injection method and Ca2+ salt of SEP-RM (Ca-SEP-RM) rhamnolipid. For the example here, the lipid concentration in ethanol was about 40 mg / mL, the ratio of ethanol solution to final volume upon injection was 1 :20, unbuffered water was used, and the time to complete the injection was about 40 seconds. The resultant rhamnosomes were subject to Dynamic Light Scattering analysis. Initial mean particle diameter was 97 nm and PDI was 0.13. After 10 days at ambient temperature, mean particle diameter was 188 nm and PDI was 0.09.Example 28 (Prophetic): Encapsulation of Water-Soluble Actives in Rhamnosome or Rhamnolipid-Modified Liposome by Means of Aqueous Acid Injection Method

[0182] Aqueous compositions comprising water-soluble actives loaded rhamnosomes or rhamnolipid-modified liposomes free of phospholipids or with various ratios of rhamnolipid to phospholipid are prepared by means of an aqueous acid injection method, wherein a solution of acid in water is injected into a solution comprising lipid and active in water. In one example, the injection is conducted at a controlled rate into a vessel of aqueous lipid with agitation. In a second example, the injection is conducted in a flow reactor. The pH of the solution of lipid in water is at or above a pH that enables full solubility of the lipid or lipid combination and active. The fabrication method enables a high degree of efficiency in encapsulation of water soluble active and does not require the use of organic solvents.Example 29: Preparation of Rhamnosomes by Means of Aqueous Acid Injection Method

[0183] A study was conducted to evaluate the fabrication of rhamnosomes in water by means of addition of aqueous acid to an aqueous solution of SEP-RM rhamnolipid. 6.0 grams of rhamnolipid actives in 60 grams of water was prepared by dissolution of SEP-RM rhamnolipid (25.9% active) in deionized water in a 150 mL beaker. Using an overhead mechanical stirrer for mixing at ambient temperature, a solution of 0.50Naqueous H3PO4 was then added via a syringe pump at an addition rate of 0.5 mL / min. Aliquots were pulled for pH measurement and characterization by dynamic light scattering immediately upon preparation and upon aging at ambient temperature. The results are summarized in Table 8 below.

[0184] Acid addition volumes that resulted in compositions in the range of pH 5.6 to 5.0 were found to produce rhamnosome compositions that were unstable in particle size, as observed by large changes in mean particle diameter between initial preparation and 4 weeks. In contrast, acid addition to a pH of about 4.5 resulted in a rhamnosome composition with particles that slightly decreased in size over 4 weeks.Example 30: Preparation of Concentrated Rhamnosome Fluid by Means of Aqueous Acid Injection Method

[0185] To a 150 mL beaker was added 50.13 g of SEP-RM rhamnolipid (25.9% active), corresponding to 13.0 g of active rhamnolipid. Using an overhead mechanical stirrer for mixing at ambient temperature, 20.9 mL of a solution of 1.0N aqueous H3PO4 was then added via a syringe pump at an addition rate of 0.5 mL / min to afford a fluid with a pH of 4.5 and rhamnolipid content of about 18%. During acid addition, the rhamnolipid composition became thick and exhibited strong shear-thinning behavior. As the composition approached pH 4.5, a rapid onset of sharp viscosity reduction was observed. The final product obtained was a hazy, low viscosity fluid. Dynamic light scattering analysis indicated a mean particle size of 235 nm and a PDI of 0.26. Aging of the product at ambient temperature for 13 weeks and re-characterization indicated a mean particlesize of 263 and PDI of 0.29, indicating good particle stability. Product viscosity at 13 weeks was measured to be 233 cP at 10 sec-1 shear rate.Example 31 : Preparation of Concentrated Lipid Fluid Comprising 70 / 30 Rhamnolipid and Soy PC by Means of Aqueous Acid Injection Method

[0186] To a 150 mL beaker was added 53.56 g of SEP-RM rhamnolipid (25.9% active) and 5.95 g of soy phosphatidyl choline (95%, Avanti Polar Lipids). The lipids were mixed for 5 hr at ambient temperature to afford a clear, homogeneous solution. Then, 24 mL of a solution of 1 .ON aqueous H3PO4 was then added via a syringe pump at an addition rate of 0.5 mL / min to afford a fluid with a pH of 4.1 and a total lipid content of about 23.7%. The final product obtained was an opaque, low viscosity fluid. Dynamic light scattering analysis indicated a mean particle size of 363 nm and a PDI of 0.25. Aging of the product at ambient temperature for 13 weeks and re-characterization indicated a mean particle size of 399 and PDI of 0.24. Product viscosity at 13 weeks was measured to be 223 cP at 10 sec-1shear rate.

[0187] The embodiments and examples described here are illustrative, and do not limit the presently described technology in any way. The scope of the present technology described in this specification is the full scope defined or implied by the claims. Additionally, any references noted in the detailed description section of the instant application are hereby incorporated by reference in their entireties, unless otherwise noted

[0188] 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

What is claimed is:1 . A composition for use in preparing rhamnolipid modified liposomes, the composition comprising::(a) a rhamnolipid mixture, wherein the rhamnolipid mixture 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; 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) optionally at least one phospholipid; and(c) optionally at least one active wherein the rhamnolipid composition is in an amount of about 1 wt% to about 99 wt% by weight, based on the total weight of the composition; wherein the at least one phospholipid is in an amount of about 1 wt% to about 99 wt% by weight, based on the total weight of the composition.

2. The composition of claim 1 , wherein the rhamnolipid mixture is a solvent extraction purified rhamnolipid composition.

3. The composition of claim 1 , wherein the rhamnolipid mixture is an acidulated solvent extraction purified rhamnolipid composition.

4. The composition of claim 1 , wherein the rhamnolipid mixture comprises a silver rhamnolipid salt, potassium rhamnolipid salt, choline rhamnolipid salt, zinc rhamnolipid salt, copper rhamnolipid salt, manganese rhamnolipid salt, calcium rhamnolipid salt, magnesium rhamnolipid salt, or combination thereof.

5. The composition of claim 1 , wherein the at least one phospholipid is a phosphatidylcholine, a phosphatidylethanolamine, a phosphatidate a phosphatidylserine, a phosphosphingolipid, a soy lecithin, or combinations thereof.

6. The composition of claim 1 , wherein the at least one active is a water soluble active.

7. The composition of claim 6, wherein the at least one active is an agricultural agent, cosmetic agent, personal care agent, pharmaceutical agent, nutraceutical agent, or combination thereof.

8. The composition of claim 6, wherein the at least one active is a ceramide, a sphingoid base, a sterol, a peptide, an amino acid, a phenol, a polyphenol, a vitamin, a rhamnolipid, or combination thereof.

9. The composition of claim 1 , wherein the composition further comprises at least one sterol.

10. The composition of claim 9, wherein the sterol comprises cholesterol, 7- dehydrocholesterol, potassium cholesterol sulfate, cholesteryl succinate, 25-hydroxy-7- dehydrocholesterol, ergosterol, fucosterol, hopanoids, hydroxysteroid, phytosterols, or combinations thereof.1 1 . The composition of claim 1 , wherein the composition further comprises an edge activator.

12. The composition of claim 1 , wherein the edge activator comprises a surfactant, a polyol or combination thereof.

13. The composition of claim 12, wherein the polyol comprises glycerin or propylene glycol.

14. The composition of claim 1 , wherein the rhamnolipid mixture 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.

15. The composition of claim 1 , wherein the rhamnolipid mixture 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.

16. The composition of claim 1 , wherein the rhamnolipid mixture 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.

17. The composition of claim 1 , wherein the rhamnolipid mixture 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.

18. The composition of claim 1 , wherein the rhamnolipid mixture is in an amount of about 1 % to about 10%, based on the total weight of the composition.

19. A method for the production of a rhamnolipid modified liposome composition, wherein the method comprises providing a composition according to any one of claims 1 -18, and performing a thin film hydration procedure.

20. The method of claim 19, wherein the method further comprises an extrusion step, a size exclusion step, a pH adjustment step, or combination thereof.21 . The method of claim 19, wherein the pH of the rhamnolipid modified liposome composition is decreased after completion of the thin film hydration procedure.

22. A method for the production of a rhamnolipid modified liposome composition, wherein the method comprises providing a composition according to any one of claims 1 -18, and performing a solvent injection procedure.

23. The method of claim 22, wherein the solvent is ethanol.

24. The method of claim 22, wherein the method further comprises an extrusion step, a size exclusion step, a pH adjustment step, or combination thereof.

25. The method of claim 22, wherein the pH of the rhamnolipid modified liposome composition is decreased after completion of the solvent injection procedure.

26. A method for the production of a rhamnolipid modified liposome composition, wherein the method comprises providing a first composition according to any one of claims 1 -18, and performing an aqueous injection procedure.

27. The method of claim 26, wherein the at least one active is a water soluble active.

28. The method of claim 26, wherein the phospholipid is a soy lecithin.

29. The method of claim 28, wherein the soy lecithin is present in an amount of about 1 wt% to about 20 wt%, about 5 wt% to about 15 wt%, or about 8 wt% to about 12 wt%, or about 10 wt %, by weight of the total composition.

30. The method of claim 26, wherein the first composition is injected into an aqueous buffer having a lower pH.31 . A rhamnolipid modified liposome composition comprising:(a) a rhamnolipid mixture, wherein the rhamnolipid mixture 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) optionally at least one phospholipid; and(c) optionally at least one active; wherein the rhamnolipid composition is in an amount of about 1 wt% to 99 wt% by weight, based on the total weight of the composition; and wherein the at least one phospholipid is in an amount of about 1 wt% to about 99 wt% by weight, based on the total weight of the composition.

32. The rhamnolipid modified liposome composition of claim 31 , wherein the rhamnolipid mixture is a solvent extraction purified rhamnolipid composition.

33. The rhamnolipid modified liposome composition of claim 31 , wherein the rhamnolipid mixture is an acidulated solvent extraction purified rhamnolipid composition.

34. The rhamnolipid modified liposome composition of claim 31 , wherein the rhamnolipid mixture comprises a silver rhamnolipid salt, potassium rhamnolipid salt, choline rhamnolipid salt, zinc rhamnolipid salt, copper rhamnolipid salt, manganese rhamnolipid salt, calcium rhamnolipid salt, magnesium rhamnolipid salt, or combination thereof.

35. The rhamnolipid modified liposome composition of claim 31 , wherein the at least one phospholipid is a phosphatidylcholine, a phosphatidylethanolamine, a phosphatidate a phosphatidylserine, a phosphosphingolipid, a soy lecithin, or combinations thereof.

36. The rhamnolipid modified liposome composition of claim 31 , wherein the at least one active is a water soluble active.

37. The rhamnolipid modified liposome composition of claim 31 , wherein the at least one active is an agricultural agent, cosmetic agent, personal care agent, pharmaceutical agent, nutraceutical agent, or combination thereof.

38. The rhamnolipid modified liposome composition of claim 31 , wherein the at least one active is a ceramide, a sphingoid base, a sterol, a peptide, an amino acid, a phenol, a polyphenol, a vitamin, or combination thereof.

39. The rhamnolipid modified liposome composition of claim 31 , wherein the composition further comprises at least sterol.

40. The rhamnolipid modified liposome composition of claim 39, wherein the sterol comprises cholesterol, 7-dehydrocholesterol, potassium cholesterol sulfate, cholesteryl succinate, 25-hydroxy-7-dehydrocholesterol, ergosterol, fucosterol, hopanoids, hydroxysteroid, phytosterols, or combinations thereof.41 . The rhamnolipid modified liposome composition of claim 31 , wherein the composition further comprises an edge activator.

42. The rhamnolipid modified liposome composition of claim 41 , wherein the edge activator comprises surfactants, polyols or combinations thereof.

43. The rhamnolipid modified liposome composition of claim 42, wherein the polyol comprises glycerin or propylene glycol.

44. The rhamnolipid modified liposome composition of claim 31 , wherein the rhamnolipid mixture 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.

45. The rhamnolipid modified liposome composition of claim 31 , wherein the rhamnolipid mixture 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.

46. The rhamnolipid modified liposome composition of claim 31 , wherein the rhamnolipids mixture 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.

47. The rhamnolipid modified liposome composition of claim 31 , wherein the rhamnolipid mixture 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.

48. The rhamnolipid modified liposome composition of claim 31 , wherein the rhamnolipid mixture is in an amount of about 0.1% to about 10%, based on the total weight of the composition.

49. The rhamnolipid modified liposome composition of claim 31 , wherein the composition comprises particles having a mean particle size of from 15 nm to 800 nm.

50. The rhamnolipid modified liposome composition of claim 31 , wherein the composition comprises particles having a mean particle size of from 50 nm to 500 nm.51 . The rhamnolipid modified liposome composition of claim 31 , wherein the composition comprises particles having a mean particle size of from 60 nm to 350 nm.

52. The rhamnolipid modified liposome composition of claim 31 , wherein the composition comprises particles having a mean particle size of from 80 nm to 240 nm.

53. The rhamnolipid modified liposome composition of claim 31 , wherein the composition comprises a polydispersity index of from 0.05 to 0.9.

54. The rhamnolipid modified liposome composition of claim 31 , wherein the composition comprises a polydispersity index of from 0.05 to 0.3.

55. The rhamnolipid modified liposome composition of claim 31 , wherein the composition comprises particles having an actives loading capacity of 2-50%, alternatively 5-30%, alternatively 10-20%.

56. The rhamnolipid modified liposome composition of claim 31 , wherein the composition comprises particles having an actives loading capacity 5-30%.

57. The rhamnolipid modified liposome composition of claim 31 , wherein the composition comprises particles having an actives loading capacity of 10-20%.

58. The rhamnolipid modified liposome composition of claim 31 , wherein the composition comprises particles having an actives loading efficiency of 30-100%.

59. The rhamnolipid modified liposome composition of claim 31 , wherein the composition comprises particles having an actives loading efficiency of 50-100%.

60. The rhamnolipid modified liposome composition of claim 31 , wherein the composition comprises particles having an actives loading efficiency of 70-100%.61 . The rhamnolipid modified liposome composition of claim 31 , wherein the composition is prepared according to any one of claims 19-30.

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