A GELATION/DE-GELATION SYSTEM FOR TRANSDERMAL DELIVERY OF AN ACTIVE

INGREDIENT (OR SUBSTRATE)

FIELD OF THE DISCLOSURE

[0001] This disclosure relates to the use of polymeric molecules for the construction of a gelation/de-gelation network (or system) in one alternative, and in another alternative a gelation network (or system) with de-gelation being optional capable of enclosing, retaining and releasing substrates. The polymeric molecules may be ionic, biocompatible, biodegradable, and stable at ambient temperature or otherwise neutral conditions. In one alternative, the gelation/de-gelation network has a further capability of releasing the retained substrate upon a chemical or physical trigger event without the use of a de-gelation component. The gelation/de-gelation network may be useful for, e.g., containing and releasing biorelevant amounts of substrates, such as therapeutic pharmaceuticals, nutritional supplements or cosmetics.

BACKGROUND

[0002] Polymeric molecules such as chitosan, alginate, pectin, and cellulose have been studied for their abilities to form networks [Cejkova J. Compartmentalized and internally structured particles for drug delivery - a review. Current Pharmaceutical Design 2013; 19: 6298-6314] Since 1990s, the focus on the study of alginate and cellulose network structures [Draget Kl, Taylor C. Chemical, physical and biological properties of alginates and their biomedical implications. Food Hydrocolloids 2011 ; 25: 251-256] shifted to include their use in drug delivery. More recently, spherical particles and nanoparticles made from biocompatible polymers were used enterically or intravenously for the delivery of small molecule drugs. In addition, medical substances such as vaccines typically administered via intradermal injections can also be delivered using nanoparticle constructs [Gregory AE, Titball R, Williamson D. Vaccine delivery using nanoparticles. Front Cell Infect Microbiol. 2013 Mar 25;3:13]

[0003] Stability of substrates retained in polymeric networks, such as alginate networks, was improved by the addition of salts as stabilizers to enable the wider use of the networks. [Marquis M, et al. Microfluidics encapsulation of Pickering oil microdroplets into alginate microgels for lipophilic compound delivery. ACS Biomater. Sci. Eng. 2016; 2: 535-543] From there, many non-medical substances such as nutrient supplements and cosmetic agents can be better stabilized within a polymeric network. However, the rate of drug release and tissue-specific targeting remain areas needing improvement, which limit the use of these delivery systems.

SUMMARY

[0004] According to one aspect, there is provided a polymeric gel matrix for enclosing, retaining and releasing a substrate, said polymeric gel matrix comprising: a. at least one gelation agent, preferably in a gelation solution; and

b. optionally, at least one de-gelation agent, preferably in a de-gelation solution; said at least one gelation agent for enclosing and retaining said substrate in said polymeric gel matrix; and for release of said substrate from said polymeric gel matrix when no de-gelation agent is present;

said at least one de-gelation agent for breaking down the polymeric gel matrix resulting in the release of said substrate from said polymeric gel matrix.

[0005] In one alternative, said at least one gelation agent is selected from the group consisting of sodium alginate, calcium chloride, calcium carbonate, calcium bicarbonate, cellulose, chitosan, pectin, iron (III) salts, magnesium salts, calcium salts and combinations thereof.

[0006] In one alternative, said at least one de-gelation agent is selected from the group consisting of sodium chloride, sodium citrate, ethylenediaminetetraacetic acid (EDTA), agarase, a strong acid, an enzyme which hydrolyses alpha(1 ,4) glycosidic linkages, a cation-specific chelating agent, and combinations thereof.

[0007] In one alternative, said polymeric gel matrix is biodegradable.

[0008] In one alternative, said substrate is selected from the group consisting of an active pharmaceutical ingredient (API), antiseptic, supplemental nutrient, cosmetic agent, recreational substance, food product, mineral, dye, tracer, biological product, prophylactic, and combinations thereof.

[0009] In one alternative, said polymeric gel matrix is a transdermal delivery system.

[00010] In one alternative, said transdermal delivery system is a topical dressing.

[00011] In one alternative, said topical dressing is a wound dressing.

[00012] In one alternative, said transdermal delivery system is a transdermal patch.

[00013] In one alternative, said gelation agent is present in an amount of from about 0.1 to

30% w/w of a volume of said gelation solution.

[00014] In one alternative, when said gelation agent is:

a. sodium alginate, it is present in an amount of from about 0.5 to 10% w/w; b. calcium chloride, it is present in an amount of from 0.1 to 30% w/w; in one alternative from 10 to 30% w/w;

c. calcium carbonate, it is present in an amount of from 0.1 to 10% w/w; in one alternative from 0.5 to 3% w/w.

[00015] In one alternative, said de-gelation agent is present in an amount of from about 1.5 to

30% w/v of the de-gelation solution.

[00016] In one alternative, said de-gelation solution has a volume of from about 5 to 20 times a volume of said gelation solution.

[00017] In one alternative, when said degelation agent is:

a. sodium citrate, it is present in an amount of from about 2% to 26% w/v; in one alternative from about 5 to 10% w/v of the de-gelation solution;

b. EDTA, it is present in an amount of from about 1.5% to 15% w/v; in one alternative from about 2.5 to 12% w/v of the de-gelation solution.

[00018] According to yet another alternative, there is provided a polymeric gel matrix for enclosing, retaining and releasing a substrate, said polymeric gel matrix comprising: a. at least one gelation agent in combination with said substrate, preferably in a gelation solution; and

b. at least one de-gelation agent, preferably in a de-gelation solution;

said at least one gelation agent for enclosing and retaining said substrate in said polymeric gel matrix;

said at least one de-gelation agent for breaking down the polymeric gel matrix resulting in the release of said substrate from said polymeric gel matrix.

[00019] In one alternative, said at least one gelation agent is selected from the group consisting of sodium alginate, calcium chloride, calcium carbonate, calcium bicarbonate, cellulose, chitosan, pectin, iron (III) salts, magnesium salts, calcium salts and combinations thereof.

[00020] In one alternative, said at least one de-gelation agent is selected from the group consisting of sodium chloride, sodium citrate, ethylenediaminetetraacetic acid (EDTA), agarase, a strong acid, an enzyme which hydrolyses alpha(1 ,4) glycosidic linkages, a cation-specific chelating agent, and combinations thereof.

[00021] In one alternative, said polymeric gel matrix is biodegradable.

[00022] In one alternative, said substrate is selected from the group consisting of an active pharmaceutical ingredient (API), antiseptic, supplemental nutrient, cosmetic agent, recreational substance, food product, mineral, dye, tracer, biological product, prophylactic, and combinations thereof.

[00023] In one alternative, said polymeric gel matrix is a transdermal delivery system.

[00024] In one alternative, said transdermal delivery system is a transdermal patch.

[00025] In one alternative, said gelation agent is present in an amount of from about 0.1 to

30% w/w of volume of said gelation solution.

[00026] In one alternative, when said gelation agent is:

a. sodium alginate, it is present in an amount of from about 0.5 to 10% w/w;

b. calcium chloride, it is present in an amount of from 0.1 to 30% w/w of volume of said gelation solution; in one alternative from 10 to 30% w/w;

c. calcium carbonate, it is present in an amount of from 0.1 to 10% w/w of volume of said gelation solution; in one alternative from 0.5 to 3% w/w;

[00027] In one alternative, said de-gelation agent is present in an amount of from about 1.5 to

30% w/v of the de-gelation solution.

[00028] In one alternative, when said de-gelation agent is:

b. sodium citrate, it is present in an amount of from about 2 to about 26% w/v; in one alternative from about 5 to 10% w/v of the de-gelation solution;

c. EDTA, it is present in an amount of from about 1.5 to 15% w/v; in one alternative from about 2.5 to 12% w/v of the de-gelation solution.

[00029] In one alternative, said polymeric gel matrix further comprises at least one of a texture modifier, detergent, and combinations thereof. In a further alternative, said texture modifier comprises at least one of an emulsifier, stabilizer, plasticizer, and combinations thereof.

[00030] In one alternative, said at least one emulsifier is selected from the group consisting of albumin, L-histidine, triglyceride, oleic acid, polyglycerol polyricinoleate (PgPr) and combinations thereof.

[00031] In one alternative, said albumin is selected from the group consisting of human albumin, bovine serum albumin, and combinations thereof.

[00032] In one alternative, said at least one detergent is selected from the group consisting of

Tween 80™, Tween 20™, Span 80™, polyglycerol polyricinoleate and combinations thereof. [00033] In one alternative, said at least one stabilizer is selected from the group consisting of calcium carbonate, calcium acetate, sodium chloride, sodium hydroxide and combinations thereof.

[00034] In one alternative, said at least one plasticizer is selected from the group consisting of glycerol, calcium bicarbonate, and hydrochloric acid.

[00035] In one alternative, said polymeric gel matrix has a gelation time from about 1 second to 60 minutes. In another alternative, said polymeric gel matrix has a gelation time from about 5 seconds to 20 minutes. In another alternative, said polymeric gel matrix has a gelation time from about 5 seconds to 30 seconds. In another alternative, said polymeric gel matrix has a gelation time from about 10 minutes to 20 minutes.

[00036] In one alternative, said polymeric gel matrix has a de-gelation time from about 10 to about 300 minutes.

[00037] In one alternative, said de-gelation time is from about 10 to about 30 minutes.

[00038] In one alternative, said de-gelation time is from about 30 to about 60 minutes.

[00039] In one alternative, said de-gelation time is from about 60 to about 90 minutes for a gel matrix of thickness about 2mm.

[00040] In one alternative, said de-gelation time is from about 90 to about 180 minutes for a gel matrix of thickness about 1 cm, when the volume of de-gelation solution applied is at least about 18x the volume of the overall gelation volume, preferably about 20x the volume of the overall gelation volume.

[00041] In one alternative, said de-gelation is triggered by a physical event, chemical event or combination thereof.

[00042] In one alternative, said physical event is selected from the group consisting of removal of barrier between the gelation and de-gelation solutions (resulting in the contact of the gelation and de-gelation solutions leading to a chemical event), dehydration, applied force (including compression, piercing, pressure, and shear), and aging.

[00043] In another alternative, when there is only a gelation component present, said physical event is selected from the addition of a de-gelation (resulting in the contact of the gelation and de-gelation components leading to a chemical event), dehydration, applied force (including compression, piercing, pressure, and shear), and aging.

[00044] In one alternative, said chemical event is selected from the change in pH, change in ion strength, and change in wavelength or intensity of light. In one alternative, this results in the breakdown of the polymerized gel matrix is caused by chelating agents irreversibly binding to divalent ions from the polymer network, taking these ions away from other components of the polymerization network.

[00045] In yet another alternative, there is provided a transdermal drug delivery kit, said kit comprising:

a. a gelation component, wherein said gelation component comprises a gelation agent and a substrate retained within said gelation component; and b. a de-gelation component, wherein said de-gelation component comprises a de gelation agent;

wherein when said gelation component is in contact with said de-gelation component, upon a trigger event said de-gelation component causes de-gelation of said gelation component resulting in release of said substrate.

[00046] In yet another alternative, there is provided a process of preparing a transdermal delivery system comprising a gelation component and a de-gelation component; said process comprising:

a. preparation of a gelation component comprising enclosing a substrate in a gelation agent and forming a gel matrix;

b. preparation of a de-gelation component comprising forming a de-gelation gel matrix.

[00047] In yet another alternative, there is provided a process of preparing a transdermal delivery system comprising a gelation component, an optional de-gelation component, and a scaffold; said process comprising:

a. preparation of a gelation component comprising enclosing a substrate in a gelation agent and forming a gel matrix;

i. the gel matrix may be formed on the scaffold, when the scaffold is placed into a mold, prior to gelation. The scaffold may be made of textile (breathable or occlusive), plaster, absorbent pad, nylon networks, paraffin film, paper (including filter paper), tape, polysaccharide film or tubing. b. optionally, preparation of a de-gelation component comprising forming a de gelation gel matrix.

[00048] Textile may be breathable or occlusive, woven or non-woven, preferably non-woven, and textile includes, but is not limited to, animal fibers such as silk, wools and casein- derived textiles; plant fibers such as but not limited to canvas, hemp, cotton, flax/linen, rayon, bamboo-derivatives; synthetic fibers such as but not limited to acrylic, nylon, polyurethane, carbon-composites; and blends or mixtures thereof. [00049] In one alternative, medium or larger weave fabrics are preferred over fine weave fabrics, as the large spaces between fibers allow more space for gelation to occur, and lead to larger gel masses adhered per area of the fabric. The preferred fabric characteristics include some water repellency(but not completely water-repellent to allow attachment of the gel thereon), holds shape when dry or wet (i.e. stiffness), provides a surface, preferably an even surface, for uniform gelation, and no fraying at edges and corners when adhered onto a polymeric gelation matrix.

[00050] In yet another alternative, there is provided a method of releasing a substrate on the epidermis or an opening on the epidermis of a patient in need of treatment; said method comprising contacting said epidermis or an opening on the epidermis of said patient with a transdermal delivery system described herein and effecting a trigger event as described herein to trigger de-gelation of a gelation component of said transdermal delivery system.

[00051] According to one aspect, there is provided a composition to create a mesh or matrix structure using at least one polymer. Such matrix structure has the ability to encapsulate at least one substrate at the time of production, and retain the at least one substrate during further processing and storage. At the time of required need where the at least one substrate needs to be released from the matrix structure, the release of the at least one substrate may be triggered through a change in the external environment to fully or partially degrade the matrix structure. Examples of gelation and de-gelation formulations, the methods of production, the methods of breakdown, and use thereof are also described here.

[00052] The polymeric composition comprises two main portions, one being at least one gelation agent, and the other being at least one de-gelation agent.

[00053] In another alternative, the polymeric composition comprises one portion being at least one gelation agent, and optionally a second portion, the second optional portion being at least one de-gelation agent.

[00054] As used herein, the term gelation means an agent that facilitates formation of a gel matrix (polymeric network) and enclosing and retaining a substrate in said gel matrix.

[00055] As used herein, the term de-gelation means and agent that facilitates breaking down of said gel matrix (polymeric network) resulting in the release of said substrate from said polymeric gel matrix.

[00056] In one alternative, the gelation agent includes, but is not limited to, sodium alginate, modified alginate including alginate covalently-bound to polyacrylamide, calcium chloride, calcium carbonate, calcium bicarbonate, cellulose, chitosan, pectin, iron(lll) salts, magnesium salts, calcium phosphate, other calcium salts. The degelation agents include, but are not limited to sodium citrate, ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis(beta-aminoethyl ether)-N,N,N’,N’-tetra acetic acid (EGTA), agarase, a strong acid, an enzyme which hydrolyses alpha(1 ,4) glycosidic linkages, a cation-specific chelating agent, and combinations thereof.

[00057] One advantage of this composition is that it is biosafe in the amounts presented, and biodegradable so that there is minimal harm to the environment or end user during composition breakdown.

[00058] In one alternative, methods of making the gel structure generally include, but are not limited to, pressure or shear homogenization (automated or manual), manual mixing, multichannel microfluidics constructs, polystyrene needle junctions, and other mixing techniques which are familiar to those skilled in the art. Once the gel structure reaches a consistent texture, the entire solution or parts of the solution may be molded into a desired form using techniques that include, but are not limited to, (1) a scaffold, such as but not limited to, filter paper wetted with one part of the gelation agent with substrate by dipping the filter paper in the gelation agent with substrate, and then dipping the filter paper or other scaffold previously dipped in the gelation agent with substrate into a medium containing the other part of the gelation agent (“Filter paper Method”); (2) pouring onto a surface the gelation agent with substrate to create layers or slabs of certain thickness that contain the gelation agent with substrate (“Pour-on Method”); (3) droplet dispersion of one part of the gelation agent with substrate, into a medium containing the other part of the gelation agent (“Mixing Method”); and (4) using dialysis tubing where one part of the gelation agent with substrate is contained inside the tubing, and a medium containing another part of the gelation agent is outside the tubing (“Dialysis tubing Method”); (5) using a horizontal gel-on-textile gelation method where one part of the gelation agent with substrate is pre-mixed into a liquid in a first container, and the other part of the gelation agent is pre-mixed into a liquid in a second container, a scaffold is floated horizontally over one part of the gelation agent in the first container, then over the second (“Horizontal Gel-on-Textile Method”).

[00059] The speed of gelation may be manipulated by varying the method of gelation, varying the mixing or shear rate, varying concentrations of each of the gelation agents, varying temperature, varying pH, or by adding a gelation inhibitor (including short chain alcohols such as methanol {1-10%}, ethanol {1-50%}; short chain ketones such as acetone; excess amounts of fatty acids, and other organic compounds and solvents). These gelation inhibitors are added during production of the gel to delay the gelation process, and may be removed by solvent evaporation to allow gel formation.

[00060] In another alternative where the gel matrix encapsulates one or more substrates, the substrate is held and retained inside the gel, in spaces or“pockets” between filaments in the gelation matrix formed by ionic, covalent, or van der waals bonding between components of the gelation portion (See Fig. 1 below). These“pockets” are able to close during gelation, and securely hold the substrate until a trigger event for de gelation or content release occurs.

[00061] In one alternative where the gel matrix is used as a component in a transdermal delivery system, the gel matrix may be used to contain an active pharmaceutical ingredient (API), antiseptic, supplemental nutrient, cosmetic agent, recreational substance, food product, mineral, dye, tracer, biological product, prophylactic, and combinations thereof.

[00062] In one alternative the gelation system may be used to package active pharmaceutical ingredients without elevating the processing temperatures during gelation and setting, minimizing modification and heat breakdown of the pharmaceutical ingredients during processing. Further, this gelation system may be used to maintain a tight internal structure with the“pockets” as described, which may hold the encapsulated substrate in place during elevated temperatures and moisture conditions, to extend the active life of the substrate.

[00063] In one alternative, the gelation system is structurally stable, and therefore may be stored, between about -30°C to about 75°C, in another alternative, between about - 20°C to about 55°C. and yet in one alternative, the gelation system may be stored between 4°C to 55°C.

[00064] In another alternative, said de-gelation agent further comprises a degradation and/or denaturing agent to facilitate degradation and/or denaturing of an active ingredient enclosed and retained in said polymeric gel matrix, wherein said active ingredient is toxic and/or harmful if released into the environment and/or if said active ingredient results in irritation of the epidermis or an opening on the epidermis if contacted with the epidermis or an opening on the epidermis for an extended period of time, to provide the additional benefit of minimizing unintentional exposure to the API during handling and disposal by healthcare practitioners or patient caretakers. For medical substances with side effects when administered systemically, such as last-resort antibiotics, a local administration near the target site may minimize some of the adverse long-term effects (such as the development of antibiotic resistance).

[00065] In another alternative, said polymeric gel matrix is stable from about pH 3-8.5, and would de-gelate at a pH lower than about 1.

[00066] In another alternative, said polymeric gel matrix is formed on a textile scaffold, and can be folded between about 10 to more than about 100 times when stored at room temperature before disintegration or content leakage. In another alternative, between about 50 to more than 100 times. A folding test can be used to determine the polymerization efficacy and physical strength of the polymer patch (i.e. polymeric gel matrix on a textile scaffold). This test was adapted from Singh and Bali (Singh A., and Bali A. Journal of Analytical Science and Technology. 2016. 7-25.). Folding endurance is defined as the number of folds required to break the polymeric gel matrix. The tested patch that is cut into a 2x2cm square was held horizontally flat manually or on a device, and is repeatedly folded in one single direction where the opposing edges touch, then returned to the original flat position after each fold. All folds should align along the same axis of folding. The number of folds until the polymeric gel matrix cracked, disintegrated, or leaked was recorded as the“folding score”. The maximum number of times a sample was folded was 100, for patch samples that did not break within 100 folds, their folding score was recorded as 100. At least 3 patches were tested for each sample condition.

[00067] In another alternative, said polymeric gel matrix is formed by the dialysis tubing method and not backed by a scaffold, and can be folded between about 2 to about 100 times when stored at room temperature before disintegration or content leakage. In one alternative, the polymeric gel matrix can be folded between about 2 to about 60 times. In another alternative, the polymeric gel matrix can be folded between about 60 to about 100 times.

[00068] In another alternative, the polymeric gel matrix can be combined with a microneedles- based transdermal delivery system such that the gel matrix is found adjacent or above the microneedles array, and the gel matrix content can be released to fill the microneedles before they are delivered through the skin by the microneedles piercing the skin of a human or animal subject. In such a combined set up, the polymeric gel matrix can be used to control the rate of substrate release into the microneedles device, and into the human or animal subject. [00069] According to another alternative, there is provided a polymeric gel matrix wherein between about 30-52% of the substrate is released over 4 hours and between about 78-93% of substrate is released over 24 hours.

[00070] According to yet another alternative, there is provided a polymeric gel matrix wherein said substrate has a rate of diffusion into skin of about 27% in 4 hours and about 41% in 24 hours.

[00071] According to yet another alternative, there is provided a polymeric gel matrix having substrate storage stability of at least 28 days at room temperature and at freezing temperatures.

[00072] According to yet another alternative, there is provided a polymeric gel matrix of any one of claims 1-42, 46-50 having substrate storage stability of at least 96 hours at 50°C.

[00073] Other aspects, features and advantages will be apparent from the following disclosure, including the detailed description, and the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[00074] Figure 1 depicts a schematic of the polymeric gel matrix according to one alternative.

[00075] Figure 2 depicts a cross section of a transdermal patch with the polymeric gel matrix according to one alternative.

[00076] Figure 3 depicts a cross section of a transdermal patch with an adhesive plaster backing, textile scaffold, and no de-gelation agents according to one alternative.

[00077] Figure 4 depicts the polymeric gel matrix at various stages in time with/without de gelation agents according to one alternative.

[00078] Figure 5 depicts the elution of a substrate that is a biologically relevant molecule from a polymeric gel matrix, compared to elution of a polymeric gel matrix without the substrate.

[00079] Figure 6 depicts the polymeric gel matrix formed by pouring technique according to one alternative.

[00080] Figure 7 depicts a microscopic view of the polymeric gel matrix according to one alternative.

[00081] Figure 8 depicts the polymeric gel matrix of Figure 2 under microscope.

[00082] Figure 9 depicts the polymeric gel matrix of Figure 2 under microscope undergoing de-gelation due to aging (34 days). [00083] Figures 10A-C depict the polymeric gel matrix formed by the dialysis tubing technique according to the alternative illustrated by Figure 11.

[00084] Figure 1 1 depicts a dialysis tubing technique to form the polymeric gel matrix according to one alternative.

[00085] Figure 12 depicts the moisture loss from polymeric gel matrices formed by the dialysis tubing method (referred to as Dialysis) and the horizontal gel-on-textile (referred to as GoT) method, respectively, over time.

[00086] Figure 13 depicts the moisture loss from polymeric gel matrix formed by the dialysis tubing method under various storage temperatures over time.

[00087] Figure 14 depicts the moisture loss from polymeric gel matrices formed by the horizontal gel-on-textile method either with or without textile scaffolding and/ or adhesive plaster backing.

[00088] Figure 15 depicts the release of a substrate that is a dye, from polymeric gel matrices formed by the dialysis tubing method and the horizontal gel-on-textile method, respectively, over time.

[00089] Figure 16 depicts the microscopic structure and substance retention levels of the double emulsion polymeric gel matrix formed by the horizontal gel-on-textile method, before and after use as a transdermal delivery path ex-vivo.

[00090] Figure 17 depicts the microscopic sectional view of the polymeric gel matrices of

Figure 6, stored under room temperature or freezing (-20°C) conditions respectively, at day 0 and day 28.

[00091] Figure 18 depicts the level of a dye delivered into a skin tissue from the polymeric gel matrices, when it is either encapsulated within an emulsion within the gel matrix (referred to as double emulsion) or directly within the gel matrix (referred to as single emulsion). Both samples were formed by the dialysis tubing method and used a transdermal delivery patch to model the delivery into skin ex-vivo.

[00092] Figure 19 depicts the polymeric gel matrix with a dye encapsulated within double emulsion structures, under microscope during storage without de-gelation agents, at ambient and elevated temperatures, over a period of time, according to one alternative.

[00093] Figure 20 depicts the release of a substrate, B12, over a period of time, that is a biologically relevant molecule from a polymeric gel matrix formed by the dialysis tubing method. [00094] Figure 21 depicts the retention of activity of a substrate, alpha-amylase, that is a biologically relevant enzyme, when embedded into the polymeric gel matrix formed by the dialysis tubing method and subject to storage at room and elevated temperatures.

[00095] The foregoing summary, as well as the following detailed description, will be better understood when read in conjunction with the appended drawings. For the purpose of illustration, there are shown in the drawings alternatives which are presently preferred. It should be understood, however, that this should not be construed as being limited to the precise arrangements and instrumentalities shown in the drawings and described in the following detailed description.

DETAILED DESCRIPTION

[00096] Referring now to FIG. 1 , there is depicted the macroscopic structure of a gelation/de gelation system with the gelation component formed by calcium-alginate polymerization. Calcium ions hold strands of the alginate molecule together in a mesh like structure, and create pockets to hold a substrate (in droplet or single molecule form) in place.

[00097] In aqueous solution, the polysaccharide strands exist as hydrocolloids. Once exposed to calcium ions, negatively charged residues bind with Ca ²⁺ ions to form links, forming a thermo- irreversible gel that remain chemically reversible. Calcium concentration correlate positively with gel strength. Calcium-alginate gels are unaffected by temperature, allowing the gelation to occur at ambient conditions.

[00098] There is also depicted a de-gelation component comprising sodium citrate and EDTA.

Depicted are the polymeric matrix 1-10 composed of gelation agents; gelation agent 1-20, in this example alginate of single strand/monomer; a substrate 1-30; an exploded view showing the alginate strand 1-31 , the optional inner phase of substrate

1-32, substrate 1-30 which may be an emulsion; and a calcium ion 1-33; and a de gelation matrix 1-34 comprising EDTA 1-35 and sodium citrate 1-36 as de-gelation agents.

[00099] Referring now to FIG. 2, there is depicted the macroscopic structure of the gelation/de-gelation system as a transdermal patch 2-10 on the epidermis 2-20. The de-gelation component 2-40 is shown, in this example, above the gelation component

2-30. The de-gelation component is in a solution in a container. The gelation component or gel matrix 2-30 is exploded to show the gelation agents crosslinked to form a network 2-31 and encapsulate the substrate. The substrate 2-32 as structured droplets, is exploded to show stabilizer molecules between the substrate and gelation agents 2-33 and the API is shown within the substrate structure 2-34.

[000100] Referring now to FIG. 3, there is depicted the macroscopic structure of the polymeric gel matrix as part of a transdermal patch on the epidermis 3-10, where the gelation portion of the matrix 3-20 is formed on a textile scaffold 3-30 and backed by an adhesive plaster backing 3-40. The adhesive plaster backing 3-40 allows for the use of the transdermal patch on the epidermis 3-10 of an animal or human substrate. In this schematic of a transdermal patch, there is no de-gelation portion.

[000101] Referring now to FIG. 4, there is depicted in the two upper frames, gel formed by calcium-alginate gelation system with vitamin B12 as substrate formed using the slab method, being stable for more than three hours at ambient storage conditions. The two lower frames depict the addition of de-gelation solution containing de-gelation agents sodium citrate and EDTA was able to degrade the calcium-alginate gelation system with vitamin B12 as substrate within three hours.

[000102] Referring now to FIG. 5, there is provided a side by side elution of the substrate, B12, which was encapsulated in the gel matrix (including sodium alginate, calcium chloride, PgPr, BSA, sodium hydroxide), and released via addition of the de-gelation agents (including sodium, calcium, EDTA, and citrate), through HPLC (left side elution) with a peak at 2.450 of the release of B12. A reference HPLC elution of the gelation and de-gelation agents (without B12 substrate) is also provided (right side elution) with a peak at 2.095. FIG. 5 clearly depicts the release of B12 from the polymeric gel without changing the structure of B12, as the B12-containing sample exhibits a higher area under the curve (AUC, 32 497 mAU/s) compared to that of the B12-free blank gel (32 309 mAU/s).

[000103] Referring now to FIG. 6, there is provided the gel matrix formed by pouring the gelation agent solution into a dish (i.e. the pour-on method) and allowing gelation to occur.

[000104] Referring now to FIG. 7, there is depicted the microscopic structure of a gel matrix of the bottom exploded portion of FIG. 2, retaining an emulsion containing Alexa Fluor® 633 dye as the substrate. Zeiss LSM 700 confocal microscope was used with C- Apochromat 40x/1.20 Water Immersion Korr M27 objective, with 639nm laser excitation at 6.5%. In the Brightfield panel, one can see the outline of the double emulsion structures within the polymeric gel. In the Laser Excitation panel, one can see the substrate (Alexa Fluor® 633 dye) encapsulated within the polymeric gel as area of fluorescence.

[000105] Referring now to FIG. 8, Alexa Fluor® 633 dye was incorporated into a double emulsion within the gelation system formed by calcium-alginate polymerization using the pour-on method. The microscopic encapsulation structure was shown to be stable for 21 days given the left and right panels depict structural stability over 21 days based on the distinct borders 8-10 surrounding the substrate. Zeiss LSM 700 confocal microscope was used with C-Apochromat 40x/1.20 Water Immersion Korr M27 objective, with 639nm laser excitation at 6.5%.

[000106] Referring now to FIG. 9, Alexa Fluor® 633 dye was incorporated into a double emulsion within the gelation system formed by calcium-alginate polymerization using the pour-on method. The microscopic encapsulation structure begins degradation by 34 days in storage at ambient conditions, as demonstrated by the non-distinct borders 9-10 around the pockets of the substrate and the gelation structure. Zeiss LSM 700 confocal microscope was used with C-Apochromat 40x/1.20 Water Immersion Korr M27 objective, with 639nm laser excitation at 6.5%.

[000107] Referring now to FIGS. 10A-C, gel matrices are formed by putting the substrate and one part of the gelation agent (including sodium alginate, calcium carbonate, sodium chloride, sodium hydroxide, and rhodamine-B as the substrate in Figure 10A; sodium chloride, sodium hydroxide, bovine serum albumin, PgPr, calcium carbonate, a lipophilic solvent, and alginate in Figure 10B; sodium chloride, sodium hydroxide, bovine serum albumin, PgPr, calcium carbonate, a lipophilic solvent, alginate, and rhodamine-B as the substrate in Figure 10C) into a dialysis tubing, and allowing formation of a gel matrix within the dialysis tubing when placed into a beaker containing the second part of the gelation solution (including calcium chloride, calcium carbonate, and a hydrophilic solvent such as water), absent the substrate.

[000108] Referring now to FIG. 11 , there is shown the substrate 1 1-10 and one part of the gelation solution 1 1-20 contained within a dialysis tubing 1 1-21 , and another part of the gelation solution 11-30 is contained in the beaker 1 1-40, absent the substrate 1 1- 10. In tubing 1 1-21 , there is also an emulsifier 11-22. The tubing 11-21 is kept sealed from the other part of the gelation solution 11-30 by an enclosure 11-23, such as a clip. Gelation solution 11-30 includes a gelation agent 1 1-31.

[000109] Referring now to FIG. 12, polymeric gel matrices were constructed using the dialysis method and the horizontal gel-on-textile (GoT) method, respectively. A 2 cm X 2 cm square of each gel was weighed immediately before and after storing at ambient temperature (20°C) for 1 , 2, 18, 24, 48, 72, and 96 hours. The reduction in mass was attributed to moisture loss due to evaporation. The reduction in mass equivalates to moisture loss due to evaporation since there is no substrate encapsulated in these gel matrices. The GoT method showed a faster rate of moisture loss initially compared to the dialysis method. However, both exhibited a similar moisture loss pattern. The moisture loss (%) was calculated for each condition with the equation: moisture loss (%) = 100 (Equation 1)

[000110] Referring now to FIG. 13, there is depicted the moisture loss from polymeric gel matrices formed by the dialysis tubing method under various storage temperatures over time. A 2x2cm square of each gel was weighed immediately before and after storing at refrigeration temperature (4°C), room temperature (20°C), or elevated temperature (55°C). Sampling timepoints included 2, 24, 48, and 72 hours. The reduction in mass equivalates to moisture loss due to evaporation since there is no substrate encapsulated in these gel matrices. The moisture loss (%) was calculated for each condition following Equation 1. Moisture loss trends at both refrigerated and heated temperature were similar indicating that with the rate of moisture loss is controlled within the polymeric gel system, irrespective of elevated storage temperature.

[000111] Referring now to FIG. 14, there is depicted the moisture loss from polymeric gel matrices formed by the horizontal gel-on-textile method either with or without textile scaffolding and/ or adhesive plaster backing at room temperature. Each gel matrix was formed into a 2 cm X 2 cm square piece. The barrier conditions involved a combination of a textile scaffold (T+ represents presence, T- represents absence) and breathable adhesive plaster backing over the scaffold or the gel directly (B+ represents presence, B- represents absence). Each gel matrix was weighed immediately before storing (t=0h) and after storage at room temperature for 72 hours. The reduction in mass was attributed to moisture loss due to evaporation. The moisture loss (%) was calculated for each condition following Equation 1.

[000112] Referring now to FIG. 15, there is depicted polymeric gel matrices formed by the dialysis tubing method, and the horizontal gel-on-textile (GoT) method respectively, with rhodamine as substrate incorporated within double emulsion structures. In-vitro diffusion study of rhodamine from the gel matrix into a receiving solution through a hydrated regenerated cellulose membrane was conducted using franz-type diffusion cells for 24 hours at 37°C, to mimic the delivery into skin tissue. Rhodamine concentration in the release medium (0.9% aqueous NaCI) was measured by fluorescence with excitation at 560 nm, emission 610 nm, at path length of 1cm. The polymeric gel matrix formed by the GoT method exhibited a faster rate of rhodamine released (51.4% released in 4 hours, and 93.0% released in 24 hours). In comparison, the polymeric gel matrix formed by the dialysis tubing method released 30.4% in 4 hours, and 78.2% in 24 hours.

[000113] Referring now to FIG. 16, there is depicted polymeric gel matrices formed using the horizontal gel-on-textile (GoT method, with Rhodamine as substrate incorporated into double emulsion structures within the gel matrix. In-vitro diffusion study of Rhodamine from the gel matrix into saline solution (receiving medium) was conducted using franz- type diffusion cells for 24 hours at 37°C. The microscopic structures were imaged before (0 hours) and after (24 hours) the diffusion study (see release results in FIG. 15). Zeiss LSM 700 confocal microscope was used with C-Apochromat 40x/1.20 Water Immersion Korr M27 objective, with 555nm laser at 1.2% intensity. Fluorescence emission was detected at 585 nm with a 580-1000 filter. The detected fluorescence level is much lower in the image after diffusion study, demonstrating significant release of the Rhodamine out of the gel matrix and into the receiving solution.

[000114] Referring now to FIG. 17, there is depicted polymeric gel matrices of Figure 15. Gel matrices via dialysis tubing method is depicted in the upper cells and horizontal gel- on-textile (GoT) method is depicted in the lower cells. The gel matrices were each stored in a closed container at either room temperature (20°C) or freezing (-20°C). Samples were collected after storage for 28 days. Thin cross sections of each gel were laid onto a microscope slide, sealed with an oil-based sealant under glass cover slip. Zeiss LSM 700 confocal microscope was used with C-Apochromat 40x/1.20 Water Immersion Korr M27 objective, with 555nm laser at varying intensity. Fluorescence emission was detected at 585 nm with a 580-1000 filter. This clearly depicts the stability of the gel matrices over 28 days at either room temperature or freezing.

[000115] Referring now to FIG. 18, there is depicted the percentage release of Rhodamine as substrate incorporated into the gel matrices within double emulsion structures (referred to as double emulsion) or directly (referred to as single emulsion) during an ex-vivo study of diffusion of substrate into animal skin. Both double and single emulsion constructed gel matrices had identical components and relative ratios of each. A rhodamine-negative (blank) gel was included as the baseline control. Depilated rat skin tissues were cleaned with 100% ethanol and wiped with a lint-free wipe to remove surface lipids and other residues. Each 2 cm x 2cm gel matrix was adhered onto an occlusive adhesive plaster backing, and placed onto a skin tissue of the same dimensions (n=3). At each sampling point (4h and 24h), rhodamine was extracted from the skin tissue using mechanical homogenization, filtered and collected, before analyzed for rhodamine content through fluorescence spectrometry using a Perkin-Elmer LS50B fluorospectrometer. The excitation wavelength was 560 nm, the emission wavelength at 610 nm, and read time 0.1s.

[000116] Referring now to FIG. 19, there is depicted polymeric gel matrix formed by the dialysis tubing method, with rhodamine within double emulsion structures as substrate. The gel matrix was then stored in sealed containers at either ambient or room temperature (20°C) or elevated temperature (50°C). Thin cross sections of each gel were laid onto a microscope slide at indicated time points, sealed with an oil-based sealant under a glass cover slip. The microscopic structures were imaged after Oh and 48h (during storage), and 96h (post-storage). Zeiss LSM 700 confocal microscope was used with C-Apochromat 40x/1.20 Water Immersion Korr M27 objective, with 555nm laser at 1.2% intensity. Fluorescence emission was detected at 585 nm with a 580-1000 filter. This shows the ability of the polymeric gel to maintain structural integrity when stored at high temperatures (i.e. 50C).

[000117] Referring now to FIG. 20, there is depicted polymeric gel matrices formed by the dialysis tubing method, with B12 incorporated within double emulsion structures as substrate. Each gel measured 2x2cm, and weighed approximately 0.5 g each, were placed into a closed container with 10ml saline solution (release medium) to allow passive diffusion. The release medium was extracted from the tube at each sampling interval (0, 2, 24, 48, and 408h) and analyzed for B12 concentration over time using high-performance liquid chromatography with C18 column. The percentage released was calculated from the concentration of B12 measured in the release medium at each sampling interval. This shows the ability of the gel matrix to retain the substrate over an extended period of time when the matrix is not called upon to release the substrate. [000118] Referring now to FIG. 21 , there is depicted polymeric gel matrices formed by the dialysis tubing method, with alpha-amylase incorporated into double emulsion structures as substrate within the gel matrix. An aqueous solution of alpha-amylase was prepared using saline as solvent to be used as baseline activity level for comparison. Each sample was stored in a closed container at either room temperature (20°C) or elevated temperature (50°C). The retained alpha-amylase activity from the samples at 1 and 24h post-storage in each sample was determined using visual colorimetric inspection. Clear color indicates retention of enzymatic activity, while darker color indicates higher loss of activity.

[000119] Tables 1A and 1 B depict the folding endurance and usability of different polymeric gel matrix set-ups. The folding test was adapted from Singh A., 2016. Folding endurance is defined as the number of folds required to break the polymeric gel matrix. The 2x2cm square tested patch was held horizontally flat manually or on a device, and repeatedly folded in one single direction where the opposing edges touch, then returned to the original flat position after each fold. All folds should align along the same axis of folding. The number of folds until the gel matrix cracked, disintegrated, or leaked was recorded as the“folding score”. The maximum number of times a sample was folded was 100. For patch set-ups that did not break within the 100 folds, their folding scores were recorded as 100. (n=3 for all matrix set-ups tested).

[000120]

Table 1A depicts the folding score of each polymeric gel matrices formed by the dialysis tubing method, the horizontal gel-on-textile method, and the pour-on method can endure before disintegration or leakage. Triplicate samples of each gel were stored at either room temperature (20°C) or freezing (-20°C) for 7 and 28 days. At the end of the storage period, the samples were subject to the fold test performed.

[000121] Table 1 B depicts the folding score and other characteristics of each type of textile when used as scaffold for polymeric gel matrices formed using the horizontal gel-on- textile method. The percentage mass of gel formed onto each types of textile scaffold was recorded as an indirect measure of gel consistency when formed onto the scaffold.

% Mass of gel Notes after fold

Textile type Notes on gel appearance

± SD (n = 3) test

Linen Rigidity: floppy when wet very wrinkled at folded area

Coverage: uneven coverage

on textile

80.9±0.6 Gel thickness: uneven

Moisture repellant: somewhat

water repellant

Textile appearance: frays at

edges

Fine weave, Rigidity: floppy when wet minimal wrinkling synthetic at folded area

Coverage: uneven coverage

on textile

76.0±1.5 Gel thickness: uneven

Moisture repellant: Very water

repellant

Textile appearance: no observed

fraying

Medium weave, Rigidity: floppy when wet least wrinkling at synthetic folded area

Coverage: even coverage on textile

84.4±0.7 Gel thickness: uneven

Moisture repellant: Easily wetted

Textile appearance: no observed

fraying Non-woven, Rigidity: stiff when wet minimal wrinkling synthetic at folded area

Coverage: even gel coverage

on textile

Gel thickness: most even gel

88.4±0.6 thickness

Moisture repellant: Moderately

wetted with water

Textile appearance: no fraying

at ends

[000122] It must be noted that as used herein and in the appended claims, the singular forms “a,”“an,” and “the” include plural references unless the context clearly dictates otherwise.

[000123] As used herein, the term“substrate” refers to any naturally occurring or synthetic molecule, compound, or particle. Preferably, a substrate is a chemical or biological compound or formulation. Examples of substrates include, but are not limited to, pharmaceutical products, food products, nutritional products, minerals, cosmetic goods, recreational substances, chemical dyes used for medical diagnosis, and other biological products, such as nucleic acids; long or short peptides; proteins; cells; antibodies; live, dead, or attenuated virus; adjuvants, small molecules; colored compounds for temporary or permanent inking; active, inactive, or partially inactive microorganisms or parts thereof; an active or inactive metabolic product of an organism; a blood sample; a living or dead organism, homogenous particle with or without any internal structure, a molecular dispersion, emulsions, solid lipid nanoparticles, multicomponent particle structures and combinations thereof. In some alternatives a substrate is a medicinal substrate, such as a therapeutic or prophylactic/preventative substrate. As used herein, the term“therapeutic substrate” refers to a substrate that has medicinal uses in treating a disease, disorder, or condition in humans or animals. Therapeutic substrates include pharmaceutical products and biological products. As used herein, the terms“prophylactic substrate” and“preventative substrate” refer to a substrate that has medicinal uses in preventing a disease, disorder, or condition in humans or animals. Prophylactic substrates include, for example, vaccines, vaccine cocktails, and immunological supplements. In another alternative, the substrate may comprise a molecular weight of from about 100 to about 1 ,400 Daltons. In another alternative, the substrate may comprise a plasmid of from about 1 to about 2,000 kb in size, or more specifically about 50-150kb in size.

[000124] As used herein, nutritional goods include, but are not limited to, consumable substances for essential physiological function or supplements optimal physiological function.

[000125] As used herein, cosmetic goods include, but are not limited to, goods used to enhance or alter physical appearance without affecting normal physiological functions or structure.

[000126] According to another aspect, there is provided a two-part solution which may gel into a polymeric matrix structure when mixed, and is biosafe and biodegradable. In one alternative, the gelation solution includes gelation agents sodium alginate (range 0.5- 10% w/w, preferably 2%), calcium chloride (range 0.1-30% w/w, preferably 10-30%), calcium carbonate (range 0.1-10% w/w, preferably 0.5-3%), with water as the solvent. In another alternative, the de-gelation solution contains de-gelation agents selected from sodium citrate (range 2-26% w/v, preferably 5-10%), and EDTA (range 1.5-15% w/v, preferably 2.5-12%), with water as the solvent. The gelation processes may be repeated multiple times depending on the gelation method, until the desired thickness or flexibility of the gel matrix is achieved.

[000127] The gel matrix may be made to various thickness and holding capacity. Factors that influence these physical attributes include, but are not limited to, variance in concentration of each of the gelation agents, variance in pH, variance in mechanical dipping or drying speed, or by adding gelation inhibitors (including short chain alcohols such as methanol {1-10%}, ethanol {1-50%}; short chain ketones such as acetone; excess amounts of fatty acids, and other organic compounds and solvents).

[000128] In one alternative, the gel matrix is from about 1 mm to 4cm thickness. In one alternative, the gel matrix is from about 1 mm to 5mm thickness. In one alternative, the gel matrix is from about 5mm to 1cm thickness.

[000129] In one alternative, the holding capacity of the gel matrix to hold a substrate is from about 0.01-10% w/v of the gelation solution.

[000130] Formation of the polymeric gel structure involves the addition of the gelation agents to the substrate. More specifically, in one alternative, the substrate may be one molecule, compound, or a more elaborate mixture of compounds. In one alternative, the substrate may be a thermally protective formulation of an active pharmaceutical ingredient (API), which may be encapsulated and retained into a flexible polymeric matrix by shear homogenization with the gelation agents.

[000131] In another alternative, the substrate may be a single or double emulsion that is composed of distinctive macroscopic layers or phases. The substrate may be processed first into droplets of single or double emulsions through a channel-based microfluidics device (references US20180098936A1 , US10195571 herein incorporated by reference), then manually mixed with the gelation agents to polymerize into a matrix structure.

[000132] In another alternative, the substrate may be a compound that is lipophilic and can diffuse through dermal tissues of a human or animal. The substrate may be mixed with half of the gelation agents and placed into a dialysis tube with smaller pores than its constituents, then placed into a water bath containing the other half of the gelation agents. Small amounts (1-5% v/v) of ethanol may be added to the solution to control the speed of gelation and therefore thickness of the gel formed. The water bath and dialysis tubing may be removed once gelation is complete.

[000133] In yet another alternative, the substrate may be a vaccine lyophilized and adsorbed onto a lint-free filter paper at specific concentration or dosage, with half of the gelation agents applied to the filter paper as well. The filter paper can then be lowered into a water bath containing the other half of the gelation agents. Polymerization will occur on the filter paper to form a gel matrix containing the dried vaccine. Filter paper can be removed once gelation is complete. The dipping method produces a film that is layered due to the series of dips alternating between the emulsion and the gelation medium while the pour method produces a thicker, less striated. The gelation processes can be repeated multiple times until the desired thickness or flexibility of the gel matrix is achieved.

[000134] In another alternative, the polymeric gel matrix may contain a mixture of substrates, such as an active pharmaceutical ingredient (i.e. vaccine) and a small colored chemical compound which may act as a temporary dye on the skin of an animal or human subject. The diffusion rates of the two substances can be manipulated to allow the same diffusion depth in the skin, which enables the dye to act as an indicator that the API has diffused to reach a certain depth or amount in the subject. In the case that a vaccine is the API, the dye can be used as indicator of successful vaccination or triggering of an immunological event. [000135] In certain alternatives, emulsifiers and/or detergents are added to the gelation agents or the de-gelation agents to give the resulting mixture the desirable consistency, stability, and texture for the specific application. Emulsifiers which may be added include, but are not limited to, human albumin, bovine serum albumin, L-histidine, triglyceride, polyglycerol polyricinoleate, oleic acid and combinations thereof. Detergents which may be added include, but are not limited to, Tween 80™, Tween 20™, Span 80™, polyglycerol polyricinoleate and combinations thereof. Both gelation and de-gelation agents can be attached onto a scaffold during making.

[000136] The gel, with or without encapsulated substrates, may be a flexible film and appear opaque, white, or transparent. In some alternatives, where the gelation agents include alginate and calcium ions, the gel structure may be cut with a sharp tool into set shapes but can withstand bending. In this alternative, the calcium-alginate matrix can embed substrates such as lipophilic droplets within the interstices of the gel network. These lipophilic droplets can further contain smaller oil or water droplets within them, which can be immiscible and contain active pharmaceutical ingredients. The larger lipophilic droplets can also be stabilized by solid particles at the interface with the polymeric gel network (FIG. 2).

[000137] In some alternatives where the gel encapsulates one or more substrates, the substrate is held and retained inside the gel, in spaces or“pockets” between the filaments in the gelation matrix formed by ionic, covalent, or van der waals bonding between components of the gelation portion (FIG. 1). These“pockets” are able to close and securely hold the substrate until the trigger event for de-gelation or content release. In the embodiments, the gel matrix is capable of holding substrates without internal structural changes or chemical interactions with the substrate for up to 12 months. Specifically, the gel matrix is capable of holding substrates without internal structural changes or chemical interactions with the substrate for 5-30 minutes, 30-90 minutes, 1-3 hours (FIG. 4), 1-3 days, 1-2 months (FIG. 8), 1-6 months, and 1-12 months.

[000138] Release of contents can occur with partial or complete degradation of the gel matrix

(FIG. 4). In the case of partial degradation, the degradation should take place on one side of the gel matrix. The degradation should occur at least to the depth of substrate encapsulation, so that the substrate can be released from the gel. In the case of complete degradation, the degradation will occur at the edge of the gel matrix, in all directions. The gel matrix will effectively become a more fluid structure, releasing the substrates from being bound and restricted in the“pockets”. In other alternatives, the release of substrate from the gel matrix can be triggered by changes in the environment including, but not limited to, breakage of barrier between gelation and de-gelation agents, changes in local pH; ionic gradient; concentration gradient; light conditions; dehydration, applied force, aging or a combination of factors thereof, and thus can be controlled to occur at a specific rate.

[000139] In some alternatives, de-gelation may occur between 10 to 300 minutes, depending on the exact concentration of gelation and de-gelation agents/ methods used, the external environment (moisture, pH, pressure). More specifically, the de-gelation may occur between 10-30 minutes, 30-60 minutes, 60 to 90 minutes for a gel matrix of thickness of about 2mm, and 90 to 180 minutes for a gel matrix of thickness of about 1 cm.

[000140] In certain alternatives, where the gel encapsulates substrates, and where pH is critical to triggering substrate release by the gel, common titration solutions such as sodium hydroxide, hydrochloric acid, calcium hydroxide, acetic acid, and citrate acid can be used to manipulate the pH of the degelation portion of the formulation.

[000141] In certain alternatives, where the gelation agents are alginate and calcium, and the substrate is an oil emulsion with an inner water phase, the gel can be resistant to melting under high temperatures. However, its degradation can take other routes, such as liquification by chemical means. Degelation agent consisting of chelating agents, EDTA, and citrate (solubilized in water) can sequester divalent cations, such as calcium and magnesium. Specifically, the mechanism of degelation consists of the chelating agents binding to the calcium ions and removing them from the carbonyl rich calcium-alginate junction zones. With calcium no longer available to form a cross link between adjacent carbohydrate chains of the alginate strands, the gel structure is destabilized. Without the gel to provide steric separation to the substrate, the encapsulated API is released into the surrounding environment (i.e. skin tissue) along with the water phase used to dissolve it. Meanwhile, the oil phase may separate from the aqueous phases but have properties in aiding the diffusion of the API into skin tissue.

[000142] In another alternative, the gelation and de-gelation agents are GRAS (generally regarded as safe), or are widely used in the food, pharmaceutical, or cosmetics industries. Human health implications and environmental fate of all components in the gelation and degelation compositions disclosed here are minimal, as at least 85% of the composition is non-toxic at non-extreme levels. The amounts used in the formulation are generally well below the threshold limit for accidental oral exposure. For example, the daily allowable oral intake of PgPr as specified by the FDA is 7.5 mg/kg.

[000143] In another alternative, in the case where the substrate or its components may be harmful or toxic to the environment when released (such as antibiotics, dyes, or other chemical treatment agents), or in the case where the substrate or its components may cause irritation when left for extended time periods on the skin (such as steroids and antihistamine), there is an option of incorporating an additive into the degelation solution to allow for neutralization, degradation, or denaturation of the substrate or its components after the gelation system is used, and prior to disposal of the gel matrix post-use. In one alternative, this is applicable in the case of a medical transdermal patch.

[000144] The release, and rate thereof, of the substrate from the gel matrix can be determined via analytical methods such as light microscopy, confocal microscopy, transmission electron microscopy, diffusion or dialysis tests followed by high-performance liquid chromatography. The rate of release is correlated with degree of degradation of the gel by degelation agents. In some circumstances, to enhance visualization and resolution during analysis, water-soluble or oil-soluble dyes of small molecular size can be incorporated into the gel matrix or the substrate. In some alternatives, where the released substrates would immediately be absorbed by a membrane or an animal skin tissue, analytical testing methods can be extended to include diffusion cells or diffusion chamber permeation tests, and histology.

[000145] In certain alternatives, where resistance against microorganisms, bacteria, virus, or fungal agents is critical, small to trace amounts of at least one antimicrobial/ antifungal agent may be added to the gelation portion of the composition. Antimicrobial/ antifungal agents include, but are not limited to, silver nanoparticles and antibiotics (such as penicillin, amoxicillin, ampicillin, kanamycin, streptomycin, vanacomycin, lineolid, and docosanol).

[000146] Potential applications include the encapsulation of preventative and therapeutic pharmaceutical products, dyes or markers for medical diagnostic procedures, nutritional supplements, cosmetics, and recreational goods.

[000147] In certain alternatives, the polymeric gel matrix is used to contain active pharmaceutical ingredients, dyes or markers for medical diagnostic procedures, supplementary nutrients, cosmetic agents, or recreational substances. Preferably, a substrate as used herein has a shelf life between 2 weeks to 10 years, has a half-life between 12 hours to 2 years, and is stable at ambient to room temperature. According to one aspect, encapsulating or surrounding a substrate in the polymeric gel matrix improves storage stability, tolerability to thermal changes, resistance to moisture and bacterial damage, and diffusivity to epidermal and dermal tissues.

[000148] In further alternatives, there is provided a transdermal patch that contains the active pharmaceutical ingredients, dyes or markers for medical diagnostic procedures, supplementary nutrients, cosmetic agents, and recreational substances. In the case of the patch, the gel matrix would be produced to be a rectangular prism, with long lengths and widths, and short height/ thickness. The gel matrix would also be adhered to an adhesive film, bandage, woven and/or non-woven fabric or surface which can be attached to the skin of a human or animal. Optionally, another film can be attached on the opposite side of the adhesive plaster backing and the gel prism, to preserve the adhesion of the plaster backing and the moisture of the gel prior to use. The patch can stay on the skin of a human or animal for a time period of between 30 minutes to 1 month. More specifically, patch can stay for 30-60 minutes, 1-5 hours, 5-24 hours, or 1-30 days. The inclusion of textile scaffold and/or adhesive plaster backing may aid in the unidirectional secretion of substrate from the polymeric gel matrix into skin (i.e. away from the textile or adhesive plaster backing).

[000149] In one alternative where the polymeric gel matrix is used to contain preventative and therapeutic pharmaceutical products in the form of an adhesive transdermal patch, the substrates can include, but are not limited to, attenuated viruses, inactivated viruses, bacterial toxoids, nucleic acids, polypeptides, proteins, anesthetics, corticosteroids, anti-histamines, antibiotics, antiseptics, and anti-inflammatory substances.

[000150] In another alternative, where the substrate is an immunologically relevant API, the polymeric gel matrix can be used as a transdermal patch to deliver the API into the dermal and subdermal tissue layers, where Langerhans cells and other types of immune cells (such as lymphocytes) reside in order to enable immunological cascades against the immunologically relevant API.

[000151] In another alternative, where the substrate is an API with immediate local action, such as an antimicrobial or cell toxin, the polymeric gel matrix can be used as a transdermal patch to deliver the API into an area of open or in-tact skin to enable the immediate local action of the API.

[000152] In one alternative where the polymeric gel matrix is used to contain dyes or markers for medical diagnostic procedures in the form of an adhesive transdermal patch, the substrates can include, but are not limited to local dyes for ultrasound, magnetic resonance imaging, and computed tomography scan. Such patches would be used prior to the diagnostic scans or procedures, and enable quick, quantitative delivery of dyes to a restricted local area of the body.

[000153] In one alternative where the polymeric gel matrix is used to contain nutritional supplements in the form of an adhesive transdermal patch, the substrates can include, but are not limited to, vitamins and minerals, natural or synthetic hormones (such as melatonin, estrogen, or progesterone), and protein supplements.

[000154] In one alternative where the polymeric gel matrix is used to contain cosmetic goods in the form of an adhesive transdermal patch, the substrates can include, but are not limited to, water absorbing materials, oil absorbing materials, caffeine, brightening agents, collagen, keratin, botulinum toxin, and hydration materials.

EXAMPLES

[000155] Example 1 : Creation of a B12-containing gelation and de-gelation system using slab method 2.65mg vitamin B12 (BioShop, Canada) was dissolved into 10ml aqueous solution containing 4.45mg bovine serum albumin as emulsifier, 190mg sodium chloride and 0.102mg sodium hydroxide as stabilizers. This solution was shear homogenized and dispersed as droplets into 30ml lipophilic solvent containing 20mg PgPr as detergent. The oil solution containing aqueous phase B12 was then dispersed into one part of the gelation solution (50ml), which included 2% w/w sodium alginate and 3% w/w calcium carbonate using high-shear homogenization. 0.5ml of the resultant liquid mixture was poured into a petri dish containing the other part of the gelation solution, which is a fine layer of calcium chloride (0.14g) spread across the bottom of the petri dish to allow gelation through the slab method. The formed calcium-alginate gel matrices post-sectioning (dimensions are 1x1x0.2cm) are presented in FIG. 4 (Oh).

[000156] As illustrated in FIG. 4, the gels formed are stable at room temperature and ambient conditions for up to 3 hours in distilled water, whereas the gel structure would dissociate and breakdown when submerged in de-gelation solution for 3 hours. In the experiment shown in Figure 4, 10ml de-gelation solution (approximately 20x the volume of the resultant gel matrix) containing 0.52g sodium citrate and 0.29g EDTA was used to submerge 5 pieces of the sectioned gel. De-gelation occurred within 3 hours without any additional mixing of the solutions, and HPLC confirmed that B12 was released into the solution from the gel with results presented in FIG. 5. The release of B12 can be seen from the elution with a minor overlapping peak at approximately 2.4 minutes. A reference HPLC elution was provided for the gelation and de-gelation agents (without B12 substrate). Although there are background solvent signals with a peak at 2.0 minutes, this peak can be differentiated from that of the B12.

[000157] Example 2: Creation of an Alexa Fluor® 633-containing gelation and de-gelation system using pour-on method

[000158] 1 pi Alexa Fluor® 633 (Thermofisher, USA) was dissolved into 10ml aqueous solution containing 4.45mg bovine serum albumin as emulsifier, 190mg sodium chloride and 0.102mg sodium hydroxide as stabilizers. This solution was shear homogenized and dispersed as droplets 30-50pm in diameter, into 20ml linoleic acid containing 14mg PgPr as detergent. The oil solution containing the water dispersions of the Alexa Fluor® 633 dye was then mixed with one part of the gelation solution (20ml), which included 2% w/w sodium alginate and 3% w/w calcium carbonate. 0.5ml of the final solution was poured into a petri dish containing the other part of the gelation solution, which is a fine layer of calcium chloride (0.14g) spread across the bottom of the petri dish to allow gelation using the pour-on method. The formed calcium-alginate gel (FIG. 6) was sectioned vertically and sealed onto microscopy slides using PoliGrip (GSK, Canada) as the waterproof sealant. The gel samples were not fixed to allow follow-on aging experiments.

[000159] Microscopy images of the gel are presented in FIG. 7, FIG. 8 (ODays) and FIG. 9

(ODays). Zeiss LSM 700 confocal microscope was used with C-Apochromat 40x/1.20 Water Immersion Korr M27 objective, with 639nm laser excitation at 6.5%. The gel was allowed to age under ambient conditions. The internal microscopic structure of the gel was maintained for up to 21 days (FIG. 8 21 Days), and had begun to disintegrate by Day 34 (FIG. 9 34Days). Disintegration of the gel matrix is demonstrated by the blurring of boundaries between the substrate signal and gel structure. [000160] Example 3: Creation of a Rhodamine B-containing gelation and de-gelation system using the dialysis tubing method

[000161] 2.5mg Rhodamine B (SigmaAldrich, USA) was dissolved into 100ml aqueous solution containing 2.25mg bovine serum albumin as emulsifier, 90mg sodium chloride and 0.05mg sodium hydroxide as stabilizers, 12g linoleic acid, 0.24g Tween80 and 8.5mg PgPr as detergent. This solution is part one of the gelation mix. 5ml of this solution was poured into a dialysis tubing (SigmaAldrich, USA) with MW cut-off of 14,000Da, flat width of 25mm, and length of 5cm. Both ends of the dialysis tubing were enclosed with plastic tubing clippers (SigmaAldrich, USA).

[000162] A petri dish was prepared with a solution comprising of 52g calcium chloride dissolved in 120ml distilled water. The dialysis tubing containing part one of the gelation mix is then placed horizontally into the petri dish to allow gelation. Gelation occurs between 3-12 minutes inside the dialysis tubing, shown in FIG. 10A.

[000163] Example 4: Creation of a uniform gelation and de-gelation polymeric gel matrix system using dialysis tubing

[000164] Following the schematic of FIG. 1 1 , 0.19g sodium chloride, 0.1 g of sodium hydroxide, and 0.5mg of bovine serum albumin was mixed into 10ml water. This first mixture was then dispersed into 25ml oil phase solution consisting mostly of canola oil, and 0.5% w/w PgPr through high-shear homogenization for 1 minute. This oil phase containing water-droplet dispersions was further dispersed into a 60ml water- based solution consisting of 3.2g sodium alginate, and 4.8g calcium carbonate through high-shear homogenization for another 2 minutes. The mixed solution (one part of the gelation solution) was filled into dialysis tubing with 12kDa cutoff and 25mm width, and submerged into another 100ml water-based solution containing 30g calcium chloride. The gelation occurred within 10 minutes of submergence, and the resultant gel had a thickness of 0.5cm and was cut into a 2x2cm square as depicted in FIG. 10B.

[000165] The weight of gels cut to dimensions of 2x2x0.5cm were measured over 96 hours to determine percentage moisture loss when stored at room temperature, as illustrated in FIG. 12 (FIG. 12 Dialysis). Furthermore, another extended study tracked the percentage moisture loss of the gel samples when stored at refrigeration temperature (4°C), room temperature (20°C), or elevated temperature (55°C) over 72 hours. Gels were stored in closed containers at respective temperatures. Results are presented in FIG. 13. The reduction in mass equivalates to moisture loss due to evaporation since there is no substrate encapsulated in these gel matrices. The moisture loss (%) was calculated for each condition following Equation 1. Sample gels of dimensions 2x2x0.5cm, with weights approximately 0.5g, were de-gelated by using 10ml de gelation solution containing 0.52g sodium citrate and 0.29g EDTA within 95-120min.

[000166] Example 5: Creation of a uniform gelation and de-gelation polymeric gel matrix system using horizontal gel-on-textile (GoT) method

[000167] 0.19g sodium chloride, 0.1mg of sodium hydroxide, and 0.5mg of bovine serum albumin was mixed into 10ml water. This first mixture was then dispersed into 25ml oil phase solution consisting mostly of canola oil, and 0.5% w/w PgPr through high- shear homogenization for 1 minute. This oil phase containing water-droplet dispersions was further dispersed into a 60ml water-based solution consisting of 3.2g sodium alginate, and 4.8g calcium carbonate through high-shear homogenization for another 2 minutes. The mixed solution (one part of the gelation solution) was placed into one petri dish (dish 1). A second petri dish was prepared containing 50ml water- based solution with 15g calcium chloride (dish 2). Squares measuring 2.1 mm on each side were cut from each textile listed in Table 1 B. The mass of each textile disc was measured weight to 0.0001 g (n = 3). The disc was first floated on the surface of the solution in dish 1 until textile appeared to be wetted completely (between 5s to 1min), then floated on the surface of the solution in dish 2 until gelation occurs to produce a uniform gel matrix of approximately 5mm thick (another 10- 15s).

[000168] The weight of gels cut to dimensions of 2x2x0.5cm were measured over 96 hours to determine percentage moisture loss when stored at room temperature, as illustrated in FIG. 12 (FIG. 12 GoT). Gels were stored in closed containers. The reduction in mass equivalates to moisture loss due to evaporation since there is no substrate encapsulated in these gel matrices. The moisture loss (%) was calculated for each condition following Equation 1. In a follow-on study (FIG. 14), gels cut to dimensions of 2x2x0.5cm were further tested for percentage moisture loss when attached to a textile scaffold or an adhesive backing, to mimic the packaging conditions for a polymeric gel matrix used as a transdermal patch. It was demonstrated that barrier conditions involving an adhesive backing on the back of a gel matrix formed using the GoT-method was most effective at reducing moisture loss at room temperature. [000169] Example 6: Comparison of polymeric gel matrices constructed using the pour-on method, the dialysis tubing method, and the horizontal gel-on-textile method.

[000170] A first solution was created by mixing 1.9g sodium chloride, 1.02mg sodium hydroxide,

5mg bovine serum albumin, and 5.6mg of rhodamine-B as substrate, into 100ml water. This first solution was then dispersed into 250ml oil phase solution consisting mostly of canola oil and 1.25g PgPr through high-shear homogenization for 1 minute. The mixture was further dispersed into a 570ml water-based solution consisting of 10.8g sodium alginate and 16.2g calcium carbonate through high-shear homogenization for another 2 minutes. This final mixture (solution 1) was separated into three portions of 190ml each, to allow for the polymerization of gel matrices using each of the three methods. A second solution containing 650ml water and 270g calcium chloride was prepared and stirred until homogeneous. This second solution (solution 2) was also separated into three portions of 300ml each.

[000171] For gel polymerization using the pour-on method, 300ml solution 2 was poured into a large petri dish, and 190ml solution 1 was slowly poured into the dish. Gelation was allowed to happen for 20 minutes to reach full polymerization state.

[000172] For gel polymerization using the dialysis tubing method, 190ml solution 1 was poured into a dialysis tubing with 12kD cutoff and 25mm flat width. Both ends of the dialysis tubing was clipped and the tubing was slowly lowered into a large tub containing the 300ml solution 2. Gelation was allowed to happen for 10 minutes before the tubing was flipped so that both sides experienced equal gelation, by allowing another 10 minutes to allow the second side to reach full polymerization state as well. The entire gelation process lasted for less than 20 minutes (a square piece cut off near the end of the dialysis tubing is shown in FIG. 10C).

[000173] For gel polymerization using the horizontal gel-on-textile method, 190ml solution 1 and 300ml solution 2 were each placed into a large beaker. Squares measuring 2.1 mm on each side were cut from a non-woven synthetic textile, and were floated first on the surface of the solution 1 until textile appeared to be wetted completely (approx. 10s), then taken from solution 1 and floated on the surface of the solution 2 until gelation occurs to produce a uniform gel matrix of approximately 5mm thick (another 10-15s). The total time of gelation for this method is between 20-25 seconds. The average thickness of the gel was 0.48 ± 0.01 mm (n = 5). The textile thickness was not included in the thickness measurement. [000174] Three polymerized gels (each with dimensions of 2x2x0.5cm and weigh approximately 0.5g) produced using each method were chosen and sealed to be stored at either room temperature or -20°C for 7 and 28 days and subject to the folding test adapted from Singh A., 2016. (Table 1A). A percentage content release study was performed on three other gels made from the dialysis tubing method, and three other gels made from the gel-on-textile method. As illustrated in FIG. 15, each gel was subject to an in-vitro diffusion study using a Franz-type diffusion cell at 37°C for over 24 hours. Franz cells with 30cm bottom diameter was lined with a hydrated regenerated cellulose membrane (12kDa cutoff) in between its bottom receiving chamber and its top donor chamber. Each rhodamine-loaded gel was cut into a circle with diameter 2.1cm and placed on top of the membrane. The system was sealed with a rubber ring and parafilm before clamped together to ensure no movement between the two chambers and the membrane in between them. The bottom receiving chamber was filled with 0.9% saline solution (Baxter, US) up to the 15ml mark before storing in the 37°C water bath. At each timepoint, the receiving solution was collected and replenished to the 15ml mark, and subject to fluorescence spectrometry using a Perkin-Elmer LS50B fluorospectrometer (Excitation 560 nm, Emission 610 nm, path length: 1 cm). Microscopic structure, as well as rhodamine availability, of the polymeric gel matrices formed using the gel-on-textile method before and after 24 hours of the Franz cell diffusion study are shown in FIG. 16. Zeiss LSM 700 confocal microscope was used with C-Apochromat 40x/1.20 Water Immersion Korr M27 objective, with 639nm laser excitation at 6.5%. The internal structures are much more dim in the post-diffusion image (right side) as its rhodamine levels were much lower than that before the diffusion study.

[000175] Meanwhile, the gels formed using the dialysis tubing method and the gel-on-textile method were also subjected to storage either at room temperature (20°C) or freezing (-20°C) for 28 days. The microscopic structures of the gels are shown in FIG.14. Zeiss LSM 700 confocal microscope was used with C-Apochromat 40x/1.20 Water Immersion Korr M27 objective, with 639nm laser excitation at 6.5%.

[000176] Gels formed using the dialysis tubing method were used for an ex-vivo diffusion study into rat skin as demonstrated in FIG. 18. Each 2x2x0.5cm gel was placed on top of a 2x2cm depilated piece of rat skin tissue, and adhered onto the skin using adhesive backing for either 4 or 24 hours. Biological triplicates were provided for each condition (n=3). At each sampling interval, the gel and adhesive were removed from the skin, and rhodamine was extracted from the skin tissue using mechanical homogenization, filtering through 40 urn nylon mesh strainer, and 1 : 1 0.9% NaCI saline solution: ethanol. The extracts were mixed and centrifuged at 18000 RCF for 10 minutes. The supernatant was separated from the pellet and analyzed for rhodamine content through fluorescence spectrometry using a Perkin-Elmer LS50B fluorospectrometer. (Excitation 560 nm, Emission 610 nm, path length: 1 cm). No-rhodamine blank polymeric gels (FIG. 18 Rho- Blank), as well as polymeric gels containing the same amount of rhodamine but through direct encapsulation instead of double emulsion (FIG. 18 Rho+ polymeric gel matrix, single emulsion) were included as reference conditions. The percentage of rhodamine extracted can be indirectly used as a measure of bioavailability of the substrate in the dermis tissue.

[000177] Additionally, the gels formed using the dialysis tubing method were subjected to storage at room temperature (20°C) or 50°C for 96 hours. The microscopic structures of the gels were imaged at 0, 48, and 96 hours as shown in FIG. 19. Zeiss LSM 700 confocal microscope was used with C-Apochromat 40x/1.20 Water Immersion Korr M27 objective, with 639nm laser excitation at 6.5%.

[000178] Example 7: Drug content analysis of a polymeric gel matrix encapsulating vitamin

B12 as substrate.

[000179] A first solution was created by mixing 0.2g sodium chloride, 0.1 Omg sodium hydroxide,

0.5mg bovine serum albumin, and 100.7mg vitamin B12 as substrate, into 10ml water. This first solution was then dispersed into 25ml oil phase solution consisting mostly of canola oil and 0.15g PgPr through high-shear homogenization for 30 seconds. The mixture was further dispersed into a 60ml water-based solution consisting of 1.1 g sodium alginate and 1.6g calcium carbonate through high-shear homogenization for 1 minute. A second solution containing 100ml water and 30g calcium chloride was prepared and stirred until homogeneous. The first solution was poured into a dialysis tubing with 12kD cutoff and 25mm flat width. Both ends of the dialysis tubing was clipped and the tubing was slowly lowered into a large tub containing the second solution. Gelation occurred within 14 minutes. The resultant gels had thicknesses between 5-7mm, and were cut into 2x2cm squares with weights between 0.4-0.55g.

[000180] Each gel was placed into a closed container with 10ml saline solution (release medium) for up to 408 hours. 1 ml was taken out of the release medium at each timepoint (2h, 24h, 48h and 408h) to analyze its B12 concentration over time using HPLC (C18 column running 30:70 methanol: water mobile phase at a rate of 0.8 mL/min). The B12 peak appeared at 6 minutes during a total run time of 7 minutes. The area under the curve at each concentration in a standard curve was used to calculate the concentration of B12 released, demonstrated in FIG. 20. The percentage released was calculated from the concentration of B12 measured at each sampling interval and the initial loading concentration in the gel, taking into account the change in volume of release medium over time.

[000181] Example 8: Thermally insulative polymeric gel matrix to maintain enzymatic activity of alpha-amylase for extended time

[000182] A first solution was created by mixing 0.1g sodium chloride, 0.5mg sodium hydroxide,

0.25mg bovine serum albumin, and 0.02g alpha-amylase (Sigma-Aldrich) as substrate, into 5ml water. This first solution was then dispersed into 10ml oil phase solution consisting mostly of canola oil and 0.6g PgPr through high-shear homogenization for 30 seconds. The mixture was further dispersed into a 25ml water- based solution consisting of 0.5g sodium alginate and 0.8g calcium carbonate through high-shear homogenization for 1 minute. A second solution containing 25ml water and 7g calcium chloride was prepared and stirred until homogeneous. The first solution was poured into a dialysis tubing with 12kD cutoff and 25mm flat width. Both ends of the dialysis tubing was clipped and the tubing was slowly lowered into a large tub containing the second solution. Gelation occurred within 10 minutes. The resultant gels had thicknesses approx. 5mm, and were cut into 2x2cm squares with weighed 0.5g. Aqueous solutions of alpha-amylase were also made by resuspending 0.02g alpha-amylase in a total of 65ml distilled water, and each aqueous solution sample consisted of an 5.5ml aliquot of the 65ml solution.

[000183] Gels were stored in sealed containers at room temperature (20°C) or 50°C for 24 hours. Samples were taken at 1 hour and 24 hours post-storage. Each gel was mechanically homogenized and resuspended in 1.5ml water, before filtration through a 40um nylon mesh. The substrate to alpha-amylase (0.8% w/w aqueous potato starch) was brought to a boil and cooled, and 0.5ml of the substrate was mixed with each gel-extract sample and allowed to react for 5 minutes. 10 uL 2.5% I2-KI was added to the reaction and mixed thoroughly. The presence of any undigested starch caused a color change from clear to violet-black, while complete starch digestion (indicating high amylase activity) caused the mixture to remain clear. FIG. 21 shows the visual observation of amylase activity at each timepoint post-storage. [000184] It will be appreciated by those skilled in the art that changes could be made to the alternatives described above without departing from the concept thereof. It is understood, therefore, that this disclosure is not limited to the particular alternatives disclosed, but it is intended to cover modifications within the spirit and scope as defined by the following Examples, test results and appended claims.