Dissolving microneedles encapsulating allergen-loaded liposomes for allergen immunotherapy

Abstract

The present disclosure generally relates to liposomes and liposomal compositions, as well as microneedle arrays comprising said liposomes or liposomal compositions, as well as skin patches comprising said microneedle arrays, as well as methods and uses of the same, relating to delivering a proteinaceous antigen to a subject and / or desensitising a subject to an allergen. Said liposomes and liposomal compositions comprise a lipid component comprising or consisting essentially of or consisting of dipalmitoylphosphocholine (DPPC), dipalmitoylphosphatidylglycerol (DPPG), and a sterol.

Description

[0001] "Dissolving microneedles encapsulating allergen-loaded liposomes for allergen immunotherapy ' '

[0002] Technical Field

[0003] The present disclosure generally relates to liposomes and liposomal compositions, as well as microneedle arrays comprising said liposomes or liposomal compositions, as well as skin patches comprising said microneedle arrays, as well as methods and uses of the aforementioned, relating to delivering a protein to a subject and / or desensitising a subject to a proteinaceous allergen.

[0004] Background

[0005] The rising prevalence of food allergies has become a global concern in recent years as they can be life-threatening. Allergen- specific immunotherapy is one treatment option showing promising outcomes of desensitization accompanied with increased clinical thresholds. However, oral immunotherapy is not recommended for highly sensitive individuals with low thresholds due to the risk of fatal reactions. Recently, there has been a growing interest in using epicutaneous immunotherapy (EPIT), which involves the repeated delivery of allergens to the skin. This approach aims to modulate the immune response by engaging Langerhans Cells and stimulating the activation of regulatory T cells. A significant challenge to improving the efficacy of EPIT is the difficulty in passing effective amounts of macromolecular allergens through the stratum corneum (SC) layer without provoking skin inflammation and irritation. A further challenge is retaining the integrity and function of the allergen from storage through to delivery. While solid microneedles for EPIT have shown promising results in increasing the efficacy of inducing allergen desensitisation in mice, in corresponding clinical trials, such solid microneedles, when loaded with higher pollen allergen concentration (>1 HEP / mL), have resulted in high numbers of patients that have tended to exhibit immediate skin reactions, amongst other adverse events.

[0006] In response to continuing safety and efficacy concerns, novel formulations for the delivery of proteinaceous allergens, such as peanut-derived allergens, are desirable.

[0007] Summary

[0008] The inventors have surprisingly found that liposomes comprising a lipid component comprising a phosphatidyl lipid (such as DPPC), a further phospholipid (such as DPPG or DOPE), and a sterol (such as stigmasterol) can be used to prepare microneedles for delivery of an allergen via the skin. The liposomes were found to exhibit responsiveness to sPLA enzyme activity and / or temperature fluctuations. Without intending to limit the scope of the present disclosure, it is believed that such characteristics make such liposomes particularly suitable for targeted skin delivery, given their affinity for the body's temperature and the presence of sPLA enzymes beneath healthy skin. Remarkably, the inventors have surprisingly discovered that dissolvable microneedles (being effective to penetrate the stratum corneum) comprising a protein (e.g. peanut allergen loaded liposomes), demonstrate substantial release rates of the encapsulated proteins (e.g. 70% of encapsulated proteins within seven hours), alongside a commendable encapsulation efficiency (e.g. 35%). The liposomes and formulations thereof were also found to exhibit robust stability for at least one month under refrigeration. A particular advantage of the liposomes is that the immunoreactivity of the released protein remains preserved, underscoring the potential of this formulation for targeted and effective skin delivery, particularly delivery across the stratum corneum layer via dissolvable microneedles.

[0009] In one aspect, the present disclosure provides for a liposome comprising a lipid component comprising or consisting essentially of or consisting of: dipalmitoylphosphocholine (DPPC); dipalmitoylphosphatidylglycerol (DPPG); and a sterol.

[0010] The sterol may any sterol, suitable examples of which include, but are not limited to stigmasterol.

[0011] The DPPC may be present in any amount, for example and without limitation, an amount of about 30% by weight to about 40% by weight, relative to the total weight of the lipid component.

[0012] The DPPG may be present in any amount, for example and without limitation, an amount of about 30% by weight to about 40% by weight, relative to the total weight of the lipid component.

[0013] The sterol may be present in any amount, for example and without limitation, an amount of about 25% by weight to about 35% by weight, relative to the total weight of the lipid component. The sterol may be stigmasterol, and present in an amount of about 25% by weight to about 35% by weight, relative to the total weight of the lipid component.

[0014] The liposome may further comprise a protein. Suitable examples of a protein include, but are not limited to, antigens. Suitable examples of antigens include, but are not limited to, peanut-derived allergens. The sterol may be stigmasterol and the protein may be a peanut-derived allergen. The sterol may be stigmasterol present in an amount of about 25% by weight to about 35% by weight, relative to the total weight of the lipid component, and the protein may be a peanut-derived allergen.

[0015] The protein may be encapsulated by the liposome.

[0016] The liposome may be of any size, for example and without limitation, the liposome may have a mean diameter (in nm) between about 120 and about 200, between about 140 and about 200, between about 130 and about 190, between about 140 and about 180, between about 140 and about 160, or between about 150 and about 170.

[0017] The liposome may have any Zeta potential, for example and without limitation, the liposome may have a Zeta potential (mv) between about 0 and about -150, between about -10 and about -100, between about -20 and about -80, for example, the liposome may have a Zeta potential (mV) between about -30 and about -40.

[0018] In a further aspect, the present disclosure provides for a liposomal composition comprising a liposome according to any aspect, embodiment, or example disclosed herein, and a polymer. The polymer may be any polymer, for example and without limitation, the polymer may be selected from the group consisting of polyvinyl alcohol, polyvinylpyrrolidone, polyethylene glycol, polylactic acid, polyglycolic acid, poly(lactic-co-glycolic acid), polycaprolactone, hyaluronic acid, chitosan, gelatin, carboxymethyl cellulose, hydroxypropyl methylcellulose, poly(y-glutamic acid), starch- based polymers, xanthan gum, alginate, dextran, pullulan, pectin, poly(2-hydroxyethyl methacrylate), polyvinyl acetate, poly aspartic acid, poly hydroxyethyl acrylate, polyhydroxybutyrate, polyhydroxyvalerate, polyethylene oxide, polypropylene fumarate, methylcellulose, hydroxyethyl starch, carrageenan, polymers of maleic anhydride, polymers of vinyl ether or methyl vinyl ether, and copolymers of monomers thereof, for example and without limitation, the polymer may be a copolymer of methyl vinyl ether and maleic anhydride, such as alternating copolymer of methyl vinyl ether and maleic anhydride, such as Gantrez AN- 139.

[0019] The lipid component of the liposome, and the polymer, may be present in the liposomal composition at any suitable concentration, for example and without limitation, the ratio of the concentration (w / v) of the polymer of the liposomal composition and the concentration (w / v) of the lipid components of the liposome in the liposomal composition, is between about 10: 1 and about 1: 10, between about 6: 1 and about 1:2, between about 6:1 and about 1: 1, between about 6:1 and about 2: 1, between about 6:1 and about 3: 1, between about 6: 1 and about 4: 1, or between about 6: 1 and about 5: 1.

[0020] In a further aspect, the present disclosure also provides for a microneedle array comprising a plurality of microneedles comprising a liposome or liposomal composition according to any aspect, embodiment or example disclosed herein.

[0021] The plurality of microneedles may be dissolvable.

[0022] The plurality of microneedles may be of any suitable shape and size, for example, the plurality of microneedles have a mean height (in pm) of between about 400 and about 1000, between about 500 and about 700, between about 550 and about 650, or between about 580 and about 620, and / or a mean base diameter (in pm) of between about 250 and about 350, or about 280 and about 300.

[0023] In a further aspect, the present disclosure also provides for a skin patch comprising a microneedle array according to any aspect, embodiment or example disclosed herein.

[0024] In a further aspect, the present disclosure also provides for a method of desensitising a subject to an allergen, the method comprising delivering: the liposome according to any aspect, embodiment or example disclosed herein; or the liposomal composition according to any aspect, embodiment or example disclosed herein, to the subject percutaneously or epicutaneously, wherein the liposome or liposomal composition comprises the allergen.

[0025] In a further aspect, the present disclosure also provides for a method of delivering an antigen to a subject, the method comprising delivering: the liposome according to any aspect, embodiment or example disclosed herein; or the liposomal composition according to any aspect, embodiment or example disclosed herein, to the subject percutaneously or epicutaneously, wherein the liposome or liposomal composition comprises the antigen.

[0026] The liposome or liposomal composition may delivered by any suitable means, for example and without limitation, the liposome or liposomal composition may be delivered using: a microneedle array, such as a microneedle array according to any aspect, embodiment or example disclosed herein; and / or a skin patch, such as a skin patch according to any aspect, embodiment or example disclosed herein.

[0027] It will be appreciated by persons skilled in the art that numerous variations and / or modifications may be made to the embodiments described herein, without departing from the broad general scope of the present disclosure. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

[0028] Brief Description of Drawings

[0029] Figure 1 depicts (A) stability test and (B) encapsulation efficiency of heat- responsive liposomes; (C) Zeta potential and sizes of sPLA responsive liposomes; and (D) TEM images of DPPC / DOPE / stigmasterol liposomes: (left) freshly prepared and (right) after 1 month of storage.

[0030] Figure 2 depicts encapsulation efficiency of (A) different proteins using DPPC / DOPE / stigmasterol and (B) liposomal formulations with cholesterol (DPPC / DOPE / chol.) or with stigmasterol (DPPC / DOPE / stig.); and (C) stability studies of liposomes.

[0031] Figure 3 depicts the cumulative release rate (RR)% of (A) quenched fluorescein from different formulation liposomes at 37 °C; (B) different formulation liposomes loaded with fluorescein-conjugated peanut protein at 37 °C; and (C) DPPC / DOPE / stigmasterol loaded with fluorescein-conjugated peanut protein at different temperatures.

[0032] Figure 4 depicts dot blot against peanut- specific rabbit IgG and human IgE and Intensity Density for Analysis of Dot blot; (B) SDS-PAGE (left) of released protein from DPPC LP-PN thermos-responsive liposomes with the removal of lipid components using 100 kDa centrifugal filters, SDS-PAGE for the release of PN protein by triton without removing the lipid components. Western blot against peanut- specific rabbit IgG (middle), and western blot against human IgE (right) of released protein from DPPC LP- PN thermo-responsive liposomes with removal of lipid components using 100 kDa centrifugal filters. M, marker; P, peanut protein; Lane (Ln) 1, heated peanut protein; Ln 2, protein released at day 1; Ln 3, protein released at day 3; Ln 4, proteins released at day 7; Ln 5, peanut protein encapsulated with liposomes; Ln 6, empty liposomes; Triton, Triton-released protein; and (C) ELISA of the released protein.

[0033] Figure 5 depicts (A) TEM of DPPG / DPPC / stigmasterol (left) and DPPC / DOPE / stigmasterol (right), scale bar = 100 nm; (B) encapsulation efficiency (EE)% of DPPG / DPPC / stigmasterol and DPPC / DOPE / stigmasterol; (C) determination of quenching concentration of fluorescein.

[0034] Figure 6 depicts (A) effect of concentration of calcium ion (0-1 OmM) on the release rate of quenched fluorescein from DPPG / DPPC / stigmasterol liposomes with and without 30 pg / mL of sPLA at 37 °C; (B) Effect of different concentrations of sPLA enzyme (0-80pg / mL) on the release rate of quenched fluorescein from DPPG / DPPC / stigmasterol liposomes with 10 mM calcium ion at 37°C; (C) The effect of stigmasterol on the sPLA2 enzyme activity as compared to cholesterol. (D) The effect of different concentrations of sPLA2 towards the release rate of peanut protein at 37 °C. The release of peanut protein is slower than the release of fluorescein, which could be due to the bonding formation between liposomes and peanut protein; (E) The release rate on the 7th hour to compare the different concentrations of sPLA2; (F) The effect of temperature (4°C, 20°C, 37°C and 50°C) on DPPG / DPPC / stigmasterol; and (G) The effect of sPLA on the release of fluorescein-conjugated peanut protein at room temperature (RT) and 37 °C..

[0035] Figure 7 depicts (A) dot blot for the determination of immunogenicity of released peanut protein. This indicates that the immunogenicity of peanut protein has been preserved; (B) SDS-PAGE, Immunoblot against IgE human serum and IgG rabbit antibodies. M: Marker, P: Crude peanut protein, sP: protein released from

[0036] DPPG / DPPC / stigmasterol with sPLA, nsP: protein released from

[0037] DPPG / DPPC / stigmasterol without sPLA, and EM: empty liposomes; and (C) Franz Diffusion Cell Test comparing the diffusion of peanut with and without liposomes.

[0038] Figure 8 depicts cytotoxicity assay (CCK-8 Assay) demonstrating that liposomes at concentration 0-200 ug / mL did not show cell cytotoxicity to (A) mouse embryonic fibroblast (MEF) cell line; and (B) colon epithelial cell (Caco2).

[0039] Figure 9 depicts (A) (left to right) Lateral and Top view: Height (600 pm) and diameter (280 pm) of microneedle tip using a bright-field microscope before insertion test; (B) Lateral view of microneedle (MN) after insertion into parafilm at 10N, 20 N, 30N, 40N and 100N: Height of microneedle tip using bright-field microscope after insertion test. Scale bar = 100pm; (C) Selection of different ratio of liposomal formulation to polymer.

[0040] Figure 10 depicts texture analyser results, different compression on the (A) reduction of microneedle length and the (B) penetrability of parafilm and (C) porcine skin; (D) Microscopic images of the holes made by the penetration on parafilm, mimic skin model and pig ear skin (from left to right). Scale bar = 100pm; (E) Lateral view of microneedle channel in mimic skin mode using brightfield microscope and the lateral view of porcine skin stained with haematoxylin and eosin (H&E) after microtome. Scale bar = 100pm; (F) Top view of microneedle channel in porcine skin captured using Scanning Electron Microscope (SEM); and (G) Top view of microneedle channel in porcine skin captured using confocal microscope z-stack.

[0041] Figure 11 depicts (left to right) lateral view and top view of fluorescently labelled peanut allergen encapsulated in liposomes in microneedle. Scale bar = 100 pm.

[0042] Figure 12 depicts (A) (left to right) TEM image of fresh liposomes, scale bar = 200nm. TEM image of liposomes with polymer, scale bar = 200 nm. TEM image of rehydrated from one-week old MN stored at 2-8 °C, scale bar = 200 nm. TEM image of rehydrated from one-month-old MN stored at 2-8 °C, scale bar = 200 nm. (b) Leakage percentage of protein from liposomes.

[0043] Figure 13 depicts (A) SDS-PAGE, western blotting against IgG rabbit antibodies and IgE human serum; M: marker, P: crude peanut protein, Lane 1, AP: Acetone- precipitated peanut protein, Lane 2, AM: Acetone -precipitated microneedle with allergen-loaded liposomes, Lane 3, AN: Acetone-precipitated 1 -month-old microneedle with allergen-loaded liposomes. (B) Dot-blot against IgG rabbit antibodies, IgE human serum and negative serum. M: marker, P: crude peanut protein, Lane 1, AP: Acetone- precipitated peanut protein, Lane 2, AM: Acetone -precipitated microneedle with allergen-loaded liposomes, Lane 3, AN: Acetone-precipitated 1 -month-old microneedle with allergen-loaded liposomes. (C) Fluorescence intensity (F.I.) of the protein; (D) Change of structure protein (%) of P versus AP; (E) Change of structure protein (%) of AP versus AM; and (f) Change of structure protein (%) of AP versus ALP, ALP: acetone- precipitated protein from liposomes.

[0044] Figure 14 depicts (A) Rat-basophil Leukemia (RBL) Assay for the investigation of the allergenicity of protein sample. AP: Acetone-precipitated peanut protein, AM: Acetone-precipitated microneedle with allergen-loaded liposomes, AN: Acetone- precipitated 1 -month-old microneedle with allergen-loaded liposomes; and (B) Cumulative release percentage of microneedle with peanut allergen-loaded liposomes, peanut allergen-loaded liposomes and peanut allergen using 3 kDa cellulose membrane, 1 mm and 2 mm porcine skin.

[0045] Figure 15 depicts (A) dissolution of micro needle after insertion in agar mimic skin under bright-field microscope; (B) (left to right) lateral view of the dissolution of microneedle after insertion in pig skin under bright field microscope for a minute, 5 minute and 10 minutes; (C) comparison of microneedle length after applying onto mimic skin model versus porcine skin over time; and (d) 6-month old microneedle tips after storing it in nitrogen-flushed aluminium sealed bag. Scale bar = 100 pm.

[0046] Detailed Description

[0047] The present disclosure describes the following various non-limiting embodiments, which relate to investigations undertaken to develop liposomes and liposomal compositions thereof, as well as microneedle arrays comprising said liposomes or liposomal compositions, as well as skin patches comprising said microneedle arrays, which can be suitable for delivering proteinaceous antigens and / or allergens to subjects.

[0048] General Terms

[0049] In the following description, reference is made to the accompanying drawings which form a part hereof, and which show, by way of illustration, several embodiments of the disclosure. It is understood that other embodiments may be utilized and structural changes may be made without departing from the scope of the present disclosure.

[0050] With regard to the definitions provided herein, unless stated otherwise, or implicitly required by the context, the defined terms and phrases include the provided meanings. Unless explicitly stated otherwise, or apparent from context, the terms and phrases below do not exclude the meaning that the term or phrase has acquired by a person skilled in the relevant art. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims. Furthermore, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0051] All publications discussed and / or referenced herein are incorporated herein in their entirety.

[0052] Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present disclosure. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present disclosure as it existed before the priority date of each claim of this application.

[0053] Throughout this disclosure, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e., one or more) of those steps, compositions of matter, groups of steps or groups of compositions of matter. Thus, as used herein, the singular forms “a”, “an” and “the” include plural aspects unless the context clearly dictates otherwise. For example, reference to “a” includes a single as well as two or more; reference to “an” includes a single as well as two or more; reference to “the” includes a single as well as two or more and so forth.

[0054] Those skilled in the art will appreciate that the disclosure herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the disclosure includes all such variations and modifications. The disclosure also includes all of the examples, steps, features, methods, processes, compounds, and compositions, referred to or indicated in this specification, individually or collectively, and any and all combinations thereof.

[0055] The term “and / or”, e.g., “X and / or Y” shall be understood to mean either “X and Y” or “X or Y” and shall be taken to provide explicit support for both meanings or for either meaning.

[0056] Unless otherwise indicated, the terms “first,” “second,” etc. are used herein merely as labels, and are not intended to impose ordinal, positional, or hierarchical requirements on the items to which these terms refer. Moreover, reference to a “second” item does not require or preclude the existence of lower-numbered item (e.g., a “first” item) and / or a higher-numbered item (e.g., a “third” item).

[0057] As used herein, the phrase “at least one of’, when used with a list of items, means different combinations of one or more of the listed items may be used and only one of the items in the list may be needed. The item may be a particular object, thing, or category. In other words, “at least one of’ means any combination of items or number of items may be used from the list, but not all of the items in the list may be required. For example, “at least one of item A, item B, and item C” may mean item A; item A and item B; item B; item A, item B, and item C; or item B and item C. In some cases, “at least one of item A, item B, and item C” may mean, for example and without limitation, two of item A, one of item B, and ten of item C; four of item B and seven of item C; or some other suitable combination.

[0058] As used herein, the term “about”, unless stated to the contrary, typically refers to a range of up to + / - 10% of the designated value, and includes smaller ranges therein, for example + / - 5% or + / - 1% of the designated value.

[0059] It is to be appreciated that certain features that are, for clarity, described herein in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any sub-combination.

[0060] Throughout the present specification, various aspects and components of the invention can be presented in a range format. The range format is included for convenience and should not be interpreted as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range, unless specifically indicated. For example, description of a range such as from 1 to 5 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 5, from 3 to 5 etc., as well as individual and partial numbers within the recited range, for example, 1, 2, 3, 4, 4.5, 4.75, and 5, unless where integers are required or implicit from context. This applies regardless of the breadth of the disclosed range. Where specific values are required, these will be indicated in the specification.

[0061] Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. The word "comprising", will be understood to include the term “consisting essentially of’, the latter term or its equivalents describing the inclusion of a stated element, integer or step, or group of elements, integers or steps, and the inclusion of any further element, integer or step, or group of elements, integers or steps, to a minimal amount, for example, an amount that does not significantly affect the precise physico-chemical properties of the remaining, stated element, integer or step, or group of elements, integers or steps. The word “consisting” as used herein will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, and the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0062] It will be appreciated that each example, aspect and embodiment of the present disclosure described herein is to be applied mutatis mutandis to each and every other example, aspect or embodiment unless specifically stated otherwise. Specific terms

[0063] As used herein, the term “subject” refers to any organism susceptible to a disease or condition that requires therapy. For example, the subject can be a mammal, primate, livestock (e.g., sheep, cow, horse, pig), companion animal (e.g., dog, cat), or laboratory animal (e.g., mouse, rabbit, rat, guinea pig, hamster). In one example, the subject is a mammal. In one embodiment, the subject is human.

[0064] As used herein, the term “treating” or “treatment” includes alleviation of any one or more symptoms associated with a specific disease or condition and reducing and / or eliminating any one or more of the symptoms.

[0065] As used herein, the term “preventing” or “prevention” includes prophylaxis of the specific disorder or condition.

[0066] As would be understood by the person skilled in the art, the liposomal compositions described herein, can be administered in a therapeutically effective amount. The term “therapeutically effective amount”, as used herein, refers to such a compound being administered in an amount sufficient to alleviate or prevent to some extent one or more of the symptoms of the disorder or condition being treated. The result can be the reduction and / or alleviation of the signs, symptoms, or causes of a disease or condition, or any other desired alteration of a biological system. For example, one result may be the reduction (e.g. a reduction in occurrence [or probability thereof] and / or severity) of one or more symptoms associated with an allergy. The term, “effective amount”, as used herein, refers to an amount of a liposome or liposomal composition, effective to achieve a desired pharmacologic effect or therapeutic improvement without undue adverse side effects. By way of example only, therapeutically effective amounts may be determined by routine experimentation, including but not limited to a dose escalation clinical trial. The term “therapeutically effective amount” includes, for example, a prophylactically effective amount. In one embodiment, a prophylactically effective amount is an amount sufficient to diminish and / or prevent an allergenic response. It is understood that “an effective amount” or “a therapeutically effective amount” can vary from subject to subject, due to variation in metabolism of the compound and any of age, weight, general condition of the subject, the condition being treated, the severity of the condition being treated, and the judgment of the prescribing physician. Thus, it is not always possible to specify an exact “effective amount”. However, an appropriate “effective amount” in any individual case may be determined by one of ordinary skill in the art using routine experimentation. Where more than one therapeutic agent is used in combination, a “therapeutically effective amount” of each therapeutic agent can refer to an amount of the therapeutic agent that would be therapeutically effective when used on its own, or may refer to an adjusted (e.g., reduced) amount that is therapeutically effective by virtue of its combination with one or more additional therapeutic agents.

[0067] The term “percutaneous” refers to something that is made, done, or effected through the skin. The term “epicutaneous” refers to something that is made, done, or effected on the skin.

[0068] The term “onset” of activity, as used herein, refers to the length of time to alleviate or prevent to some extent one or more of the symptoms of the disorder or condition being treated following the administration of the liposome or liposomal composition. The term “duration” refers to the length of time that the therapeutic continues to be therapeutically effective, i.e., alleviate or prevent to some extent one or more of the symptoms of the disorder or condition being treated. The person skilled in the art would be aware that onset, peak, and duration of therapy may vary depending on factors such as the patient, the condition of the patient, and the route of administration.

[0069] As used herein, the term “liposome” refers to any lamellar, multilamellar, or solid lipid particle, or to a vehicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers. In some embodiments, the liposome is a lipid nanoparticle, which shall be understood to refer to lipid-based particles having at least one dimension on the order of nanometers (e.g., 1-1,000 nm). Typically, a liposome as used herein can be formed by mixing one or more lipids. In some embodiments, the liposome has an volume that is encapsulated by a bilayer of lipids. The volume may be sufficient to receive and contain (entirely or at least partially) a protein of interest. In some embodiments, the liposome is an approximately spherical aggregate. The liposome may be formed by at least a double layer or bilayer wherein the layers are in close proximity to each other. The apolar regions of the lipids in each layer may be oriented towards the apolar regions of the lipids in the other layer, and thus the polar regions of phospholipids may form the inner and outer face of the double layer or bilayer. In some embodiments, the liposome may have a single bilayer. Alternatively, the liposome may have from 2 to 6 bilayers of lipids. When multiple lipid layers are present, those layers may be concentric, or alternatively, may not be concentric. The liposomes may be formulated in a composition for delivery of a protein.

[0070] As used herein, the term “sterol” refers to a hydroxyl derivative of a compound with four rings, A, B, C and D arranged in the specific configuration as shown below:

[0071] The sterol is a hydroxyl derivative, meaning that the above configuration may be substituted with a hydroxyl group at any permitted position. A sterol may be further substituted with any group, e.g., an alkyl group, such as alkyl Ci-Cio at any of the positions within the above configurations, and / or the ring system may be modified, e.g. saturated or unsaturated. Non-limiting examples of sterols include cholesterol and stigmasterol: cholesterol stigmasterol

[0072] As used herein, “DPPC” refers to dipalmitoylphosphocholine (Also known as 1,2- dipalmitoyl-sn-glycero-3-phosphocholine or 1 ,2-dipalmitoyl-L-phosphatidylcholine).

[0073] As used herein, “DOPC” dioleoylphosphatidylcholine (also known as 1,2- dioleoy 1- sn-gly cero- 3 -pho sphocholine) . As used herein, “DMPC” refers to dimyristoylphosphatidylcholine (also known as l,2-dimyristoyl-sn-glycero-3-phosphocholine or dimyristoyl-L-a- lecithin).

[0074] As used herein, “DPPG” refers to dipalmitoylphosphatidylglycerol (also known as l,2-dipalmitoyl-sn-glycero-3-phosphocholine or 1,2-Dipalmitoyl-L- pho sphatidylcholine) .

[0075] As used herein, “DOPE” refers to dioleoylphosphatidylethanolamine (also known as l,2-dioleoyl-sn-glycero-3-phosphoethanolamine).

[0076] As used herein, the term “lipid component” refers to the component(s) of a liposome (or liposome composition) that includes one or more lipids. In some embodiments, the lipid component comprises all lipids that are comprised by the liposome. The lipid component may form the outer layer (or ‘membrane’) of the liposome. Thus, the liposomes disclosed herein may be generally spherical and may comprise an outer lipid layer, such as an outer lipid bilayer.

[0077] As used herein, "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymer can comprise modified amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labelling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids such as homocysteine, ornithine, p- acetylphenylalanine, D-amino acids, and creatine), as well as other modifications known in the art. The term, as used herein, refers to proteins, polypeptides, and peptides of any size, structure, or function. Polypeptides include encoded polynucleotide products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments and other equivalents, variants, and analogs of the foregoing. A polypeptide can be a monomer or can be a multi-molecular complex such as a dimer, trimer or tetramer. They can also comprise single chain or multichain polypeptides. Most commonly disulfide linkages are found in multichain polypeptides. The term polypeptide can also apply to amino acid polymers in which one or more amino acid residues are an artificial chemical analogue of a corresponding naturally occurring amino acid. The term “proteinaceous substance” will be understood to encompass peptides, polypeptides and proteins.

[0078] As used herein, “antigen” refers to any proteinaceous substance that is recognized by the immune system as foreign or non-self, prompting an immune response. These substances can include proteins and polypeptides and they are typically found on the surface of pathogens such as bacteria, viruses, fungi, and parasites. When an antigen enters the body, it is identified by immune cells, such as B cells and T cells, which then initiate a series of responses to neutralize or eliminate the threat. The specific part of the antigen that is recognized by the immune system is known as an epitope or antigenic determinant. Antigens can also be derived from non-infectious sources, such as pollen, food proteins, transplanted tissues and organs, or intracellular proteins that are expressed or displayed on a cell surface (such as tumour antigens), which can all trigger an immune response, including an allergic reaction, or an inappropriate inflammatory reaction, such as transplant rejection. Antigens of the present disclosure include, but are not limited to, allergens. As used herein, an “allergen” is a proteinaceous substance that can trigger an immune response in certain individuals, leading to allergic reactions. These substances can be found in a variety of sources, including (but not limited to) foods, pollen, pet dander, insect stings, medications, and latex.

[0079] Liposomes

[0080] The present disclosure provides for a liposome comprising a lipid component comprising or consisting essentially of or consisting of: dipalmitoylphosphocholine (DPPC); dipalmitoylphosphatidylglycerol (DPPG); and a sterol.

[0081] In some embodiments, the lipid component comprises the DPPC, DPPG and sterol. In some embodiments, the lipid component consists of the DPPC, DPPG and sterol. In some embodiments, the lipid component consists essentially of the DPPC,

[0082] DPPG and sterol.

[0083] In some embodiments, the sterol is cholesterol, stigmasterol, or a combination thereof. In some embodiments, the sterol is cholesterol or stigmasterol. In some embodiments, the sterol is cholesterol. In some embodiments, the sterol is stigmasterol.

[0084] It is within the capabilities of the person skilled in the art to determine and formulate the components of the liposome in any desired ratio. Accordingly, there is no particular limitation as to the amount of DPPC that may be present. In some embodiments, the DPPC is present in amount of about 30% by weight to about 40% by weight, about 32% by weight to about 38% by weight, or about 34% by weight to about 36% by weight, relative to the total weight of the lipid component. In some embodiments, the DPPC is present in amount of about 35% by weight, relative to the total weight of the lipid component.

[0085] It will be appreciated by a person skilled in the art that the weight percentages described herein may refer to average weights determined in a sample of liposomes. It will also be appreciated that a person skilled in the art will be able to determine the weight components in the lipid component using known methods, such as but without limitation, by using chromatographic methods to separate the lipid components and subsequently determine the amount of each that is present, and / or the relative proportions of each.

[0086] Further, it will be appreciated that the final weight percentages of the lipid components in the liposomes can be achieved by selecting appropriate methods of constructing the liposomes, which may or may not require the selection of appropriate concentrations of different lipid solutions.

[0087] There is no particular limitation as to the amount of DPPG that may be present. In some embodiments, the DPPG is present in amount of about 30% by weight to about 40% by weight, about 32% by weight to about 38% by weight, or about 34% by weight to about 36% by weight, relative to the total weight of the lipid component. In some embodiments, the DPPG is present in amount of about 35% by weight, relative to the total weight of the lipid component.

[0088] There is no particular limitation as to the amount of sterol that may be present. In some embodiments, the sterol is present in amount of about 25% by weight to about 35% by weight, about 27% by weight to about 33% by weight, or about 29% by weight to about 31% by weight, relative to the total weight of the lipid component. In some embodiments, the sterol is present in amount of about 30% by weight, relative to the total weight of the lipid component.

[0089] In one example, the liposome comprises a lipid component comprising: DPPC in amount of about 30% by weight to about 40% by weight;

[0090] DPPG in amount of about 30% by weight to about 40% by weight; and sterol in amount of about 25% by weight to about 35% by weight, relative to the total weight of the lipid component.

[0091] The present disclosure also provides for a liposome comprising a lipid component comprising or consisting of: a phosphatidylcholine selected from dipalmitoylphosphocholine (DPPC), dioleoylphosphatidylcholine (DOPC) or dimyristoylphosphatidylcholine (DMPC), present in amount of about 30% by weight to about 40% by weight; dioleoylphosphatidylethanolamine (DOPE) present in amount of about 30% by weight to about 40% by weight; and a sterol present in amount of about 25% by weight to about 35% by weight, wherein all weights are given relative to the total weight of the lipid component. In one example, the phosphatidylcholine is DPPC. In another example, the sterol is stigmasterol. In another example, the phosphatidylcholine is DPPC and the phosphatidylcholine is stigmasterol. A particular advantage of one such example is that that greater thermo-responsiveness of the liposome is achieved.

[0092] DPPC, DOPC, DMPC, DPPG, DOPE, and sterol compounds such as cholesterol and stigmasterol are well known to persons skilled in the art. It is within the understanding and capabilities of the person skilled in the art to source DPPC, DOPC, DMPC, DPPG, DOPE, and sterol compounds either commercially from established chemical suppliers or through contract manufacturing organizations. Alternatively, such compounds can be synthesized using well-documented procedures available in the scientific literature. It will be appreciated that any of the components of the lipid component may be provided as a salt, for example, a sodium salt.

[0093] The person skilled in the art will appreciate that one or more additional lipids may be present in the liposome or liposomal composition. Suitable lipids for inclusion will be well known to the person skilled in the art. In some embodiments the liposome further comprises one or more additional lipids selected from the group consisting of: neutral lipid, structural lipid and PEGylated lipid. In some embodiments, the liposome further comprises an additional lipid selected from ionizable lipids, such as a cationic lipid, or an anionic lipid.

[0094] The term "neutral lipid" refers to any of a number of lipid species that exist either in an uncharged or neutral zwitterionic form at a selected pH. Suitable examples of neutral lipids include, but are not limited to, l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn- glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn- glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O- octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), l-oleoyl-2- cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn- glycero-3-phosphocholine (C16 Lyso PC), l,2-dilinolenoyl-sn-glycero-3- phosphocholine, 1 ,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1 ,2- didocosahexaenoyl-sn-glycero-3-phosphocholine, l,2-diphytanoyl-sn-glycero-3- phosphoethanolamine (ME 16.0 PE), l,2-distearoyl-sn-glycero-3-phosphoethanolamine, l,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, l,2-dilinolenoyl-sn-glycero-3- phosphoethanolamine, 1 ,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1 ,2- didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, l,2-dioleoyl-sn-glycero-3- phospho-rac-(l -glycerol) sodium salt (DOPG), and sphingomyelin.

[0095] Suitable examples of structural lipids include, but are not limited to, cholesterol, stigmasterol, fecosterol, sitosterol, campesterol, stigmasterol, brassicasterol, ergosterol, tomatidine, tomatine, ursolic acid and alpha-tocopherol.

[0096] It will be apparent to the skilled person that reference to a PEGylated lipid is a lipid that has been modified with polyethylene glycol. Suitable examples of PEGylated lipids include, but are not limited to, PEG-modified phosphatidylethanolamines, PEG- modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, and PEG-modified dialkylglycerols, optionally PEG-c- DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, and PEG-DSPE.

[0097] In some embodiments the liposome is anionic. By this it will be understood that that the liposome comprises at least one negatively charged moiety at a selected pH (and is therefore ionizable such that it may form at least one negatively charged moiety at a selected pH). It will be understood that various lipids within the lipid component may independently be neutral, negatively charged, or positively charged. Thus it will be understood that in some embodiments, the liposome has net negative charge. In some embodiments, the net charge of the liposome polymer is more positive (or less negative) at a pH in the range of 4-6.5 than at a pH in the range of 7-8. In other embodiments, the net charge of the liposome is more positive (or less negative) at a pH of 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4 or 6.5 than at a pH of 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8. In some embodiments, the liposome is neutral at physiological pH. In some embodiments, the liposome is negatively charged at physiological pH. In other embodiments, the liposome is positively charged at physiological pH.

[0098] As used herein “zeta potential” refers to the electrokinetic potential of lipids in a lipid composition. In some embodiments, the liposome has a Zeta potential (mV) between about 0 and about -150, between about -10 and about -100, between about -20 and about -80. In some embodiments, the liposome has a Zeta potential (mV) between about -30 and about -40. In some embodiments, the zeta potential is measured in water. In other embodiments, the zeta potential is measured in phosphate buffered saline (PBS). In other embodiments, the zeta potential is measured at physiological pH.

[0099] The liposomes of the present disclosure may further comprise any protein, peptide or polypeptide. Accordingly, in some embodiments the liposome further comprises a protein. The person skilled in the art will appreciate that the protein, peptide or polypeptide may be any such protein, peptide or polypeptide that is desired to be delivered percutaneously or epicutaneously when so comprised, and that such proteins, peptides or polypeptides will be known to persons skilled in the art, or may be determined so by any known method. In one example, the protein has a molecular weight (in kDa) about or less than about 150, 120, 100, 80, 60, 40, 20 or 10. Thus, the protein may have a molecular weight in the range of about 100 to about 20, such as about 80 to about 40, such as about 80 to about 60 kDa.

[0100] The protein, peptide or polypeptide may be derived from any source, including but not limited to, a human source, an animal source, or plant source. The liposome may comprise one, two, three, four or more types of protein. In some embodiments, the protein is an antigen. There is no particular limitation as to suitable antigens. It will be appreciated that any antigen suitable to be delivered percutaneously or epicutaneously when so comprised may be used, and that such antigens will be known to persons skilled in the art, or may be determined so by any known method.

[0101] In some embodiments, the protein is an allergen. There is no particular limitation as to suitable allergens. It will be appreciated that any allergen suitable to be delivered percutaneously or epicutaneously when so comprised may be used, and that such antigens will be known to persons skilled in the art, or may be determined so by any known method.

[0102] In some embodiments the antigen is a peanut-derived allergen. Suitable peanut- derived allergens for compounding with the liposomes of the present disclosure include, but are not limited to any protein or allergen derived from Arachis hypogaea, such as Ara h 1, Ara h 2, Ara h 3, Ara h 4 (also known as Ara h 3.02), Ara h 5, Ara h 6, Ara h 7, Ara h 8, Ara h 9, Ara h 10, Ara h 11, Ara h 12, Ara h 13, Ara h 14, Ara h 15, Ara h 16, Ara h 17, or Ara h 18. Accordingly, in some embodiments, the antigen is selected from Ara h 1, Ara h 2, Ara h 3, Ara h 4, Ara h 5, Ara h 6, Ara h 7, Ara h 8, Ara h 9, Ara h 10, Ara h 11, Ara h 12, Ara h 13, Ara h 14, Ara h 15, Ara h 16, Ara h 17, Ara h 18, or a combination thereof. In some embodiments, the antigen is selected from Ara h 1, Ara h 2, Ara h 3, Ara h 6, or a combination thereof. In some embodiments, the antigen is selected from Ara h 1, Ara h 2, Ara h 3, or a combination thereof. Protein sequences for the aforementioned Ara h allergens are known in the scientific literature, and by way of example, are disclosed in Burks et al. (1995). Recombinant peanut allergen Ara h I expression and IgE binding in patients with peanut hypersensitivity. The Journal of clinical investigation, 96(4), 1715-1721 (Ara h 1); Chatel et al. (2003). Isolation and characterization of two complete Ara h 2 isoforms cDNA. International archives of allergy and immunology, 131(1), 14-18 (Ara h 2); Rabjohn et al. (1999). Molecular cloning and epitope analysis of the peanut allergen Ara h 3. The Journal of clinical investigation, 103(4), 535-542 (Ara h 3); Kleber-Janke et al. (1999). Selective cloning of peanut allergens, including profilin and 2S albumins, by phage display technology. International archives of allergy and immunology, 119(4), 265-274 (Ara h 4, 5, 6, 7); Mittag et al. (2004). Ara h 8, a Bet v 1 -homologous allergen from peanut, is a major allergen in patients with combined birch pollen and peanut allergy. The Journal of allergy and clinical immunology, 114(6), 1410-1417 (Ara h 8); Krause et al. (2009). Lipid transfer protein (Ara h 9) as a new peanut allergen relevant for a Mediterranean allergic population. The Journal of allergy and clinical immunology, 124(4), 771-8.e5 (Ara h 9); Schwager et al. (2017). Peanut oleosins associated with severe peanut allergy -importance of lipophilic allergens for comprehensive allergy diagnostics. The Journal of allergy and clinical immunology, 140(5), 1331-1338. e8 £Ara h 10, 11, 14, 15); Petersen et al. (2015). Peanut defensins: Novel allergens isolated from lipophilic peanut extract. The Journal of allergy and clinical immunology, 136(5), 1295-301. e3015 (Ara h 12, 13); and Mattsson et al. (2021). Cyclophilin - A novel cross-reactive determinant in peanut. Clinical and experimental allergy : journal of the British Society for Allergy and Clinical Immunology, 51(4), 620-622 (Ara h 18), the entire contents of each of which, and including in particular, the amino acid sequences of the proteins or peptides referred to in each reference, including the proteins noted in the foregoing description of these references, are hereby incorporated herein by reference. Protein sequences for the aforementioned Ara h allergens are also available on a number of publicly available databases known to a person skilled in the art, including UniProt and others. The protein disclosed herein may be any protein having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with any protein or allergen derived from Arachis hypogaea, such as Ara h 1, Ara h 2, Ara h 3, Ara h 4 (also known as Ara h 3.02), Ara h 5, Ara h 6, Ara h 7, Ara h 8, Ara h 9, Ara h 10, Ara h 11, Ara h 12, Ara h 13, Ara h 14, Ara h 15, Ara h 16, Ara h 17, or Ara h 18, including such % identities to any of the specific prior art references referred to herein and incorporated herein by reference.

[0103] The protein may be encapsulated by or associated with the liposome. Thus, any reference herein to a liposome “comprising” a protein is to be understood as including that the protein may be encapsulated by or associated with the liposome. In some embodiments the protein is encapsulated by the liposome. As used herein, "encapsulation" (as well as the related term “encapsulated”) may refer to complete, substantial, or partial enclosure, confinement, surrounding, or encasement. In some embodiments, the protein is completely encapsulated in the liposomes or lipid particles. Alternatively, the protein may be partially embedded within the lipid component of the liposome, such that one or more parts of the protein may extend therefrom. That extension may be external to the liposome, or internal. The liposomes may include multiple proteins (which may be the same or different proteins) encapsulated by or associated with the liposome to any or varying extents.

[0104] Any known method may be used to form the liposome, and to compound, associate, encapsulate, surround, encase, or confine the protein, partially or completely, on, within or around the liposome. Suitable methods include, but are not limited to, those described elsewhere herein, and the thin film hydration method, for example, as described in Xiang, B., Cao, DY. (2018). Preparation of Drug Liposomes by Thin-Film Hydration and Homogenization In: Lu, WL., Qi, XR. (eds) Liposome-Based Drug Delivery Systems. Biomaterial Engineering. Springer, Berlin, Heidelberg, the entire contents of which is hereby incorporated herein by reference. The liposome (without or without a protein) may be formed with the use of any known buffer, suitable examples of which include but are not limited to, Tris, PBS or HEPES. As known by persons skilled in the art, temperature, other conditions, or stabilisers (for example and without limitation, trehalose or sucrose) may be used during the hydration process, which may assist in preserving the integrity of protein.

[0105] As used herein, “encapsulation efficiency” refers to the amount of a protein that becomes part of the liposome or liposomal composition, relative to the initial total amount of protein used in the preparation of the liposome. For example, if 92 mg of protein is encapsulated in an liposome out of a total 100 mg of protein initially provided to the composition, the encapsulation efficiency may be given as 92%. The efficiency of encapsulation of the protein within the liposome may be at least 20%, for example about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. Thus, the efficiency of encapsulation of the protein within the liposome may be at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%. In some embodiments, the encapsulation efficiency is at least 35%. In certain embodiments, the encapsulation efficiency is at least 40%.

[0106] In some embodiments, the liposomes have a mean diameter (in nm) between about 120 and about 200, between about 140 and about 200, between about 130 and about 190, between about 140 and about 180, between about 140 and about 160, or between about 150 and about 170. The diameter of the liposome may be measured by dynamic light scattering (DLS), transmission electron microscopy (TEM), scanning electron microscopy (SEM), or other methods that are known in the art.

[0107] In some embodiments, the liposomes are substantially homogenous. A polydispersity index may be used to indicate the homogeneity of the liposomes. A small, for example less than 0.3 or less than 0.2, poly dispersity index generally indicates a narrow particle size distribution. A composition of the liposomes described herein may have a polydispersity index from about 0 to about 0.25, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25. In some embodiments, the polydispersity index of the liposomes may be from about 0 to about 0.20 or from about 0.05 to about 0.20.

[0108] Any method known in the art may be used to determine the stability of the liposomes, for example and without limitation, by storing the liposome at selected temperature for a selected period of time, and comparing the change in size (i.e. diameter) of the liposome across the time period. A liposome sample may be considered stable, if after the selected period of time has elapsed, the mean diameter of the liposome within the sample either remains unchanged relative to the mean diameter immediately before the period of time began, or has changed by no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% relative to the mean diameter immediately before the period of time began. In some embodiments, the stability is determined according to any method described herein. In some embodiments, the liposome is stable for at least 7 days at a temperature of about 4 °C. In some embodiments, the liposome is stable for at least 30 days at a temperature of about 4 °C.

[0109] Any method known in the art may be used to determine the release rate of the liposome. Release rate refers to the rate of release of any component of the liposome, for example, any associated or encapsulated protein. Release rate is typically reported as a percentage of an associated or encapsulated protein that is released from the liposome after a set period of time (for example 25% after 1 hour). Release rate may depend on factors to which the liposome is responsive, for example, temperature, pH, enzyme(s), and buffer. In some embodiments, the liposome has a release rate about or greater than about 40% after 10 hours at 37 °C in the presence of phospholipase A2 (PLA2) at a concentration (in pg / mL) between about 30 and about 80. In some embodiments, the phospholipase A2 (PLA2) is present at a concentration (in pg / mL) of 30, 60 or 80. In some embodiments, the PLA2 is selected from secreted PLA2 (sPLA2), cytosolic PLA2 (cPLA2), Ca2+independent PLA2 (iPLA2), platelet-activating factor acetylhydrolase (PAF-AH) or lysosomal PLA2. In some embodiments, the PLA2 is selected from secreted PLA2 (sPLA2). In some embodiments, the secreted PLA2 (sPLA2) is porcine pancreatic sPLA2. In some embodiments, the secreted PLA2 (sPLA2) is human sPLA2. The person skilled in the art will appreciate that PLA2 may require the presence of Ca2+for activity. Accordingly, in some embodiments, the liposome has a release rate about or greater than about 40% after 10 hours at 37 °C in the presence of phospholipase A2 (PLA2) at a concentration (in jag / mL) between about 30 and about 80, and a calcium salt (for example CaCh) at a concentration of about 5 mM to 20 mM, for example 5 mM or 10 mM. In some embodiments, the buffer is Tris buffer of HEPES buffer. In some embodiments, the pH is pH 7.4.

[0110] Liposomal Compositions

[0111] The present disclosure also provides for a liposomal composition comprising a liposome according to any aspect, embodiment or example disclosed herein; and a polymer. A particular advantage of such liposomal compositions is that they may be utilised for delivery of the liposome via microneedle.

[0112] There is no particular limitation as to suitable polymers that may be included in the liposomal composition. Generally however, such polymers will be suitable for use in forming a microneedle of a microneedle array. Accordingly, in some embodiments, the polymer is selected from the group consisting of polyvinyl alcohol, polyvinylpyrrolidone, polyethylene glycol, polylactic acid, polyglycolic acid, poly(lactic-co-glycolic acid), polycaprolactone, hyaluronic acid, chitosan, gelatin, carboxymethyl cellulose, hydroxypropyl methylcellulose, poly(y-glutamic acid), starch- based polymers, xanthan gum, alginate, dextran, pullulan, pectin, poly(2-hydroxyethyl methacrylate), polyvinyl acetate, poly aspartic acid, poly hydroxyethyl acrylate, polyhydroxybutyrate, polyhydroxyvalerate, polyethylene oxide, polypropylene fumarate, methylcellulose, hydroxyethyl starch, carrageenan, polymers of maleic anhydride, polymers of vinyl ether or methyl vinyl ether, and copolymers of monomers thereof.

[0113] In some embodiments, the polymer is a copolymer of methyl vinyl ether and maleic anhydride, optionally an alternating copolymer of methyl vinyl ether and maleic anhydride. In some embodiments, the polymer is a Gantrez™-type polymer, which is a copolymer of maleic anhydride with methyl vinyl ether, in which the anhydride moiety may be, although need not be, in a partially or fully hydrolysed or alcoholised form. In some embodiments, the polymer is a Gantrez AN polymer. In some embodiments, the polymer is Gantrez AN- 139. Thus, the polymer may be a synthetic, alternating copolymer of methyl vinyl ether and maleic anhydride, which may or may not be sold under the trade name Gantrez™ AN- 139. The polymer may be that identified by CAS No: 9011-16-9, having a linear formula (CyHsO^n or (C4H2O3 CsHeOln, according to the following chemical structure: wherein n may be any integer such that the molecular weight of polymer is between about 200000 Da and about 2000000 Da, for example about 1000000 Da.

[0114] There is no particular limitation as to the ratio at which the liposome according to any aspect, embodiment, or example disclosed herein; and the polymer, are provided in the liposomal composition. The person skilled in the art will appreciate that a liposomal composition according to the present disclosure may be formed, for example and without limitation, by combining a liposome or the components thereof (which may be provided in a solution of known or determinable concentration) with a polymer (which may be provided in a solution of known or determinable concentration).

[0115] The concentration of the polymer in the liposomal composition may be about or less than about 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, 3 or 1.5% (w / v). In some embodiments, the concentration of the polymer is between about 1% and about 55%, between about 1% and about 50%, between about 1% and about 40%, between about 1% and about 30% between about 1.5% and about 10%, between about 1.5% and about 5%, between about 1.5% and about 3%, between about 5% and about 30%, between about 5% and about 20%, between about 5% and about 15%, between about 5% and about 20%. Examples of a liposomal composition having a polymer present in a concentration between about 1% and about 55%, or about 10%, have been found to facilitate greater loading capacity.

[0116] The concentration (in % w / v) of the lipid components of the liposome in the liposomal composition may be about or greater than about 0, 0.025, 0.05, 0.075, 0.1, 0.125, 0.15, 0.175, 0.2, 0.225, 0.25, 0.275, 0.3, 0.325, 0.35, 0.375, 0.4, 0.425, 0.45, 0.475, 0.5, 0.525, 0.55, 0.575, 0.6, 0.625, 0.65, 0.675, 0.7, 0.725, 0.75, 0.775, 0.8, 0.825, 0.85, 0.875, 0.9, 0.925, 0.95, 0.975, or 1. In some embodiments, the concentration (in % w / v) of the lipid components of the liposome is between about 0.025 and about 1, between about 0.05 and about 0.4, or between about 0.2 and about 0.3.

[0117] The concentration (in mg / mL) of the lipid components of the liposome of the liposomal composition may be about or greater than about 0, 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5, 5.25, 5.5, 5.75, 6, 6.25, 6.5, 6.75, 7, 7.25, 7.5, 7.75, 8, 8.25, 8.5, 8.75, 9, 9.25, 9.5, 9.75, or 10. In some embodiments, the concentration (in mg / mL) of the lipid components of the liposome is between about 0.25 and about 5, between about 0.5 and about 4, between about 1 and about 3, or between about 2 and about 3.

[0118] The ratio of the concentration (w / v) of the polymer of the liposomal composition and the concentration (w / v) of the lipid components of the liposome of the liposomal composition, may be about or less than about 10: 1, 9: 1, 8: 1, 7: 1, 6: 1, 5: 1, 4: 1, 3: 1, 2: 1, 1: 1. The ratio of the concentration (w / v) of the polymer of the liposomal composition and the concentration (w / v) of the lipid components of the liposome of the liposomal composition, may be about or greater than about 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2 or 1: 1. The ratio of the concentration (w / v) of the polymer of the liposomal composition and the concentration (w / v) of the lipid components of the liposome of the liposomal composition, may be in a range provided by any two of the previously described upper and / or lower amounts, for the example, the ratio of the concentration (w / v) of the polymer of the liposomal composition and the concentration (w / v) of the lipid components of the liposome of the liposomal composition may be between about 10: 1 and about 1: 10, between about 6: 1 and about 1:2, between about 6: 1 and about 1: 1, between about 6:1 and about 2: 1, between about 6:1 and about 3: 1, between about 6:1 and about 4: 1, or between about 6: 1 and about 5: 1.

[0119] The present disclosure also provides for a liposomal composition comprising a liposome according to any aspect, embodiment or example disclosed herein, and a pharmaceutically acceptable carrier. Such liposomal compositions may be formulated for administration via any accepted mode of administration for lipid particles including LNPs, liposomes, lipid vesicles and like lipid-based particles. In general terms, by “carrier” is meant a solid or liquid fdler, binder, diluent, encapsulating substance, emulsifier, wetting agent, solvent, suspending agent, coating or lubricant that may be safely administered to any subject, e.g., a human. Depending upon the particular route of administration, a variety of acceptable carriers, known in the art may be used, as for embodiment described in Remington's Pharmaceutical Sciences (Mack Publishing Co. N.J. USA, 1991). Such compositions may be formulated into preparations in solid, semisolid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suspensions, suppositories, injections (including via microneedle), inhalants, gels, microspheres, and aerosols. Typical routes of administering such liposomal compositions include, without limitation, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal, and intranasal. The compositions administered to a subject may be in the form of one or more dosage units, where for example, a tablet or injectable liquid volume may be a single dosage unit. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example, see Remington: The Science and Practice of Pharmacy, 20th Edition (Philadelphia College of Pharmacy and Science, 2000), the entire contents of which is hereby incorporated herein by reference. Upon formulation, compositions of the present disclosure will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically / prophylactically effective. The dosage ranges for the administration of the liposomes are those large enough to produce the desired effect.

[0120] Preparation methods for the above liposomal compositions are described further herein and / or are known in the art. For example, independent solutions of the polymer and liposome may be mixed at a predetermined ratio that results in the resultant liposomal composition having the liposome and / or polymer present at any of the upper described concentrations.

[0121] Microneedle array

[0122] The present disclosure also provides for a microneedle array comprising a plurality of microneedles comprising the liposome or liposomal composition according to any aspect, embodiment, or example provided herein.

[0123] Microneedles and microneedle arrays, as well as methods for their manufacture and use are well known in the art, for example as described in Chakraborty et al. ‘Current Status of Microneedle Array Technology for Therapeutic Delivery: From Bench to Clinic.’ Mol Biotechnol (2023), the entire contents of which is hereby incorporated herein by reference. Microneedles assist to penetrate the stratum corneum of the skin and remain in the skin during delivery of the protein. Microneedles are frequently situated in the form of an array, for example a 19x19 array. The array may be any size suitable for application to a subject. The microneedles have a porous structure and / or a central channel in order to be passable to the protein, and / or are dissolvable. Such arrays are discussed, for example, in US6,908,453, and in US2006 / 030812 and US2005 / 261632 and US8,834,423, the entire contents of which are hereby incorporated by reference. Accordingly, in some embodiments, the plurality of microneedles are dissolvable. By this it will be understood that the microneedle is capable of dissolving following penetration of the skin while remaining in the skin, and thereby improve delivery (e.g. delivery rate) of the drug. In some embodiments, the plurality of microneedles exhibits a dissolution rate of about or less than about 30, 25, 20, 15 or 10 minutes.

[0124] The person skilled in the art will appreciate that a microneedle, which in some instances may be approximately conical in shape, may be defined in terms of its height and base diameter. Thus, in some embodiments, the plurality of microneedles has an mean height (in pm) of between about 400 and about 1000, between about 500 and about 700, between about 550 and about 650, or between about 580 and about 620. In some embodiments, the plurality of microneedles has a mean base diameter (in pm) of between about 250 and about 50, or about 280 and about 300.

[0125] The amount of protein loaded in the microneedle array may determined by any suitable means known to the person skilled in the art, including the methods described herein, for example, by encapsulating a fluorescently labelled protein, and using a spectrofluorometer. Loading capacity is defined as the amount of protein present in the microneedle tip, and may be calculated by multiplying the concentration used with the volume of liposomal formulation.

[0126] In some embodiments, the microneedle array has a loading capacity of at least 3 pg. The microneedle array may have a loading capacity (in pg) between about 1 and about 4000, between about 1 and about 2000, between about 1 and about 1000, between about 1 and about 500, between about 1 and about 250, between about 1 and about 200, between about 1 and about 150, between about 1 and about 100, between about 1 and about 90, between about 1 and about 80, between about 1 and 70, between about 1 and about 60, between about 1 and about 50, between about 1 and about 40, or between about 1 and about 30. Each of the plurality of microneedles may have an average loading capacity (in pg) of between 0.01 and about 3, between about 0.01 and about 2, between about 0.01 and about 1, between about 0.01 and about 0.50, between about 0.01 and about 0.40, between about 0.01 and about 0.30, between about 0.10 and about 0.20, or between about 0.15 and about 0.20.

[0127] In the context of microneedle formation, encapsulation efficiency is defined as the amount of protein incorporated in liposomes detected in the microneedle over the amount of protein incorporated in liposomes that were added to the microneedle. Such encapsulation efficiency is known to persons skilled in the art and may be determined by any suitable method, for example, by encapsulating a fluorescently labelled protein, and using a spectrofluorometer. In some embodiments, the microneedle array has a encapsulation efficiency (in %) greater than about 50, 55, 60 or 65.

[0128] Delivery efficiency refers to the amount of protein from a microneedle or microneedle array that is delivered to a subject through the skin as a percentage of the protein in the microneedle or microneedle array prior to delivery. It may be determined by any suitable method known in the art including those described herein. It will be appreciated that delivery does not necessarily occur instantaneously with administration of the microneedle array, thus the delivery efficiency may be stated in reference to a period of time through which delivery has occurred (e.g. 20% after 1 minute), or alternatively, in reference to the maximum delivery efficiency achievable after an indeterminate amount of time, after which no further increase in delivery efficiency is determinable. In some embodiments, the microneedle array has a delivery efficiency (in %) greater than about 50, 55, 60, 65, 70, 75, 80 or 85, after 1, 2, 4, 6 or 8 hours. In some embodiments, delivery efficiency is stated with reference to 1 mm porcine skin. In some embodiments, delivery efficiency is stated with reference to 2 mm porcine skin. In some embodiments, delivery efficiency is stated with reference to human skin.

[0129] It will be appreciated that where the protein loading and delivery efficiency of the microneedle array is known, the amount of delivered protein will be determinable by the person skilled in the art. Thus, in some embodiments, the microneedle array is capable of delivering about or greater than about 1.0, 1.5, 2.0 or 2.5 pg of the protein to human skin. The microneedle array may be capable of delivering an amount of protein (in pg) to the human skin between about 1 and about 4000, between about 1 and about 2000, between about 1 and about 1000, between about 1 and about 500, between about 1 and about 250, between about 1 and about 200, between about 1 and about 150, between about 1 and about 100, between about 1 and about 90, between about 1 and about 80, between about 1 and 70, between about 1 and about 60, between about 1 and about 50, between about 1 and about 40, or between about 1 and about 30, or between about 1 and about 10, or between about 1 and about 5. Each of the plurality of microneedles may be capable of delivering an amount of protein (in pg) to the human skin between about 0.01 and about 3, between about 0.01 and about 2, between about 0.01 and about 1, between about 0.01 and about 0.50, between about 0.01 and about 0.40, between about 0.01 and about 0.30, between about 0.10 and about 0.20, or between about 0.15 and about 0.20.

[0130] In some embodiments, the plurality of microneedles are homogeneous or substantially homogeneous. By this it will be understood that the density and / or concentration of protein-loaded liposomes is are homogeneous or substantially homogeneous throughout the plurality of microneedles.

[0131] Any known method for microneedle array manufacture may be used to form the microneedle array of the present disclosure. Suitable methods may include, but are not limited to, those described elsewhere herein, and those discussed in Faraji et al. An overview of microneedle applications, materials, and fabrication methods. Beilstein J Nanotechnol. 2021 Sep 13;12: 1034-1046, the entire contents of which are hereby incorporated by reference herein. Typically, a liposomal composition comprising the liposome and the polymer are mixed and transferred to a mould defining the microneedle array and allowed to set. Optionally, prior to setting, the solution within the mould may be placed under reduced pressure (e.g. to remove any trapped air within the solution), and optionally any step may be repeated to ensure complete filling of the mould with the liposomal-polymer composition. A polymeric ‘backing’ may be added to the mould to provide further stability, which is typically a polymer solution (which may be the same or a different polymer to that used in the liposomal composition). Any suitable material may be used to form the backing, for example and without limitation, polymers including those described elsewhere herein, including copolymers of maleic anhydride and methyl vinyl ether, PVA or PCL. Such polymer solution may be at a higher polymer concentration to the polymer concentration of the liposomal composition. After setting of the microneedle array, it may be removed from the mould. Thus, in some embodiments, the microneedle array further comprises a backing. In some embodiments, the concentration of the polymer in the backing is twice that of the polymer within the liposomal composition comprised by the microneedle array.

[0132] The microneedle array may be formed on or mounted on a skin patch, which may aid application of the array to a subject. Accordingly, the present disclosure also provides for a skin patch comprising a microneedle array according to any aspect, embodiment, or example disclosed herein. In use, the microneedle array (optionally comprised by a skin patch) is applied directly to the skin such that the microneedles penetrate the skin. A force is typically applied to the back of the microneedle array in order to facilitate penetration. Where comprised by a skin patch, the patch may be fixed on the surrounding skin surface to hold the array in place, e.g. by action of an adhesive pre-applied to the patch, or by using medical adhesive tape. The microneedle array remains applied to the skin for a time sufficient to deliver an acceptable and / or effective amount of the liposomes comprised within.

[0133] Methods and Uses

[0134] The liposomes, liposomal compositions, microneedle arrays and skin patches disclosed herein may be administered to subjects by any suitable means of administration, which includes transdermal delivery via microneedle. It will be appreciated that the liposomes, liposomal compositions, microneedle arrays and skin patches may be used to achieve a variety of desired medical outcomes, including treatment or prevention of a variety of diseases or conditions. Accordingly, there is no particular limitation as to diseases or conditions which may be treated by use of the liposomes, compositions, microneedle arrays or skin patches disclosed herein, and the person skilled in the art will appreciate that the liposomes may comprise and / or encapsulate any protein that is desired to be used in the treatment or prevention of a given disease or condition, and that such treatment or prevention may be effected by delivery of the liposomes via any method, e.g. a microneedle array comprising the liposomes. It will be understood that this includes, but is not limited to, desensitisation of a subject towards the delivered protein, which may for example, be an allergen. Thus, in one, non-limiting example, the condition may be an allergy.

[0135] Accordingly, the present disclosure also provides for a method of desensitising a subject to an allergen, the method comprising delivering: the liposome according to any aspect, embodiment, or example disclosed herein; or the liposomal composition according to any aspect, embodiment, or example disclosed herein, to the subject percutaneously or epicutaneously, wherein the liposome or liposomal composition comprises the allergen.

[0136] The present disclosure also provides for a method of delivering an antigen to a subject, the method comprising delivering: the liposome according to any aspect, embodiment, or example disclosed herein; or the liposomal composition according to any aspect, embodiment, or example disclosed herein, to the subject percutaneously or epicutaneously, wherein the liposome or liposomal composition comprises the antigen.

[0137] As used herein, “delivery” (or the related term “delivering”) refers to the act or manner of transporting a protein, liposome, liposomal composition to, through or via an intended destination.

[0138] As used herein, "desensitising" or “desensitise” refers to a process by which the likelihood and / or severity of an immune response of a subject to an allergen is reduced or modified. One example of how this may occur includes through controlled and repeated exposure to the allergen, in order to diminish the subject's sensitivity to the allergen, and thereby lower the likelihood or severity of an allergic reaction upon subsequent exposures. It will therefore be appreciated that while in some instances, repeated doses of e.g. a liposome comprising an allergen according to the present disclosure, may be required in order to achieve a reduction in the subject’s immune response towards an allergen, that any single dose thereof should be contemplated as an act which desensitises the subject towards that allergen.

[0139] In some embodiments, the liposome or liposomal composition is delivered using: a microneedle array; and / or a skin patch.

[0140] In some embodiments, the liposome or liposomal composition is delivered using a microneedle array. In some embodiments, the liposome or liposomal composition is delivered using a microneedle array according to any aspect, embodiment, or example disclosed herein. In some embodiments, the liposome or liposomal composition is delivered using a skin patch. In some embodiments, the liposome or liposomal composition is delivered using a skin patch according to any aspect, embodiment, or example disclosed herein.

[0141] In some embodiments, delivery of the liposome or liposomal composition comprises applying the microneedle array or skin patch to a portion of the subject’s skin. It will be understood that once the microneedle array or skin patch has been applied to the skin, thereby penetrating the skin with the microneedles, that the microneedle array or skin patch may be retained or held in place for a period of time to allow delivery of the liposome from the microneedle, for example, by dissolution of the microneedle.

[0142] In some embodiments, delivery of the liposome or liposomal composition comprises applying a force (in N) of about or greater than about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 to the microneedle array or skin patch.

[0143] In some embodiments, the method comprises treatment of an allergy. In some embodiments, the method comprises treatment of a food allergy. In some embodiments, the method comprises treatment of a peanut allergy. In some embodiments, the method comprises treatment of an allergy to an allergen selected from Ara h 1, Ara h 2, Ara h 3, Ara h 4, Ara h 5, Ara h 6, Ara h 7, Ara h 8, Ara h 9, Ara h 10, Ara h 11, Ara h 12, Ara h 13, Ara h 14, Ara h 15, Ara h 16 or Ara h 17. In some embodiments, the method comprises treatment of an allergy to an allergen selected from Ara h 1, Ara h 2, Ara h 3, or Ara h 6. In some embodiments, the method comprises of an allergy to an allergen selected from Ara h 1, Ara h 2, or Ara h 3.

[0144] The present disclosure also provides for a use of a liposome or liposomal composition according to any aspect, embodiment, or example disclosed herein, in the densensitising of a subject to an allergen, wherein the liposome or liposomal composition comprises the antigen.

[0145] The present disclosure also provides for a use of a liposome or liposomal composition according to any aspect, embodiment, or example disclosed herein, in the delivery of an antigen to a subject, wherein the liposome or liposomal composition comprises the antigen.

[0146] The present disclosure also provides for a use of a liposome or the liposomal composition according to any aspect, embodiment, or example disclosed herein, in the manufacture of a medicament for the densensitising of a subject to an allergen, wherein the liposome or liposomal composition comprises the antigen. The present disclosure also provides for a use of a liposome or liposomal composition according to any aspect, embodiment, or example disclosed herein, in manufacture of a medicament for the delivery of an antigen to a subject, wherein the liposome or liposomal composition comprises the antigen.

[0147] In some embodiments, the liposome or liposomal composition is to be delivered or formulated to be delivered percutaneously or epicutaneously.

[0148] The present disclosure also provides for a liposome, liposomal composition, microneedle array or skin patch according to any aspect, embodiment, or example disclosed herein, for use in desensitising a subject to an allergen, wherein the liposome or liposomal composition comprises the allergen.

[0149] The present disclosure also provides for a liposome, liposomal composition, microneedle array or skin patch according to any aspect, embodiment, or example disclosed herein, for use in delivering an antigen to a subject, wherein the liposome or liposomal composition comprises the antigen.

[0150] ***

[0151] The present disclosure can also be described by reference to one or more of the following non-limiting experimental materials, methodologies and examples. It will be appreciated that the specific examples presented below are not intended to be limiting to the scope. It will be appreciated that persons skilled in the art may incorporate one or more of the elements or features in the examples below with those of any aspect, embodiment or example described herein to form combinations not explicitly set forth herein. All such embodiments are considered to be within the scope of the disclosure.

[0152] Examples

[0153] Materials

[0154] 1 ,2-dipalmitoyl-sn-glycero-3-phospho-( 1 ’ -rac-glycerol) (DPPG), 1 ,2-dipalmitoyl-sn- glycero-3-phosphocholine (DPPC), l,2-distearoyl-sn-glycero-3-phosphoethanolamine- poly(ethylene glycol) (DSPE-PEG), l,2-dioleoyl-sn-glycero-3-phsophoethanolamine (DOPE) and stigmasterol were obtained from Avanti Polar Lipids. Stock lipid solution of DPPG (50 mg / mL) was prepared by dissolving in chloroform and adding a small amount of methanol (2 % v / v) and MilliQ water (1 % v / v) to aid in solubilising the long chain, saturated acidic DPPG. Stock lipid solutions of DPPC and stigmasterol (each at 50 mg / mL) were devised by dissolving the compound in chloroform. Phospholipase A2 (PLA2, > 600 units / mg protein) from porcine pancreas was purchased from Sigma and was dissolved in phospholipase buffer (5mM Tris-HCl, lOmM CaCh at pH 7.4). Peanut protein was freshly made using Bundy Holt Peanut by first de-skinning the peanut, grinding the peanut using a coffee grinder and defatting it using n-hexane. Extraction of protein uses 0.1M Tris-HCl buffer mixed with 0.9% NaCl at pH 8.4. NHS-fluorescein (5,6- carboxyfluorescein succinimidyl ester) and phosphate buffer saline (PBS) pH7.4 were acquired from Sigma. NHS-fluorescein was solvated in dimethylformamide (DMF) at a concentration 50 mg / mL and diluted using PBS to a desired concentration. Gantrez AN- 139 was obtained commercially and the polydimethylsiloxane (PDMS) mould was made (as is within the capabilities of the person skilled in the art). Example 1 - Optimisation of Liposomes

[0155] Method - Preparation of Liposomes

[0156] Lipid vesicles were prepared according to the thin film hydration method as mentioned in Xiang, B., Cao, DY. (2018). Preparation of Drug Liposomes by Thin-Film Hydration and Homogenization In: Lu, WL., Qi, XR. (eds) Liposome-Based Drug Delivery Systems. Biomaterial Engineering. Springer, Berlin, Heidelberg, the entire contents of which is hereby incorporated herein by reference. Briefly, an appropriate amount of lipid stock solution (DPPG / DPPC / stigmasterol, 1.2 / 1.2 / 1 [v / v / v], or DPPG / DSPE- PEG / stigmasterol, 1.2 / 1.2 / 1, [v / v / v] or DPPG / DOPE / stigmasterol or DPPG / DPPC / cholesterol, or the preceding formulations without a sterol) was pipetted into a glass vial and dried overnight to form a thin film layer. The thin film was hydrated with protein-conjugated with NHS -fluorescein in PBS solution (50 pg / mL) at a temperature above its phase transition temperature (45 °C). To form protein-conjugated with NHS -fluorescein, briefly, 50 mg / mL NHS-5,6 carboxyfluorescein was dissolved in DMSO. A molar ratio of 1:20 of protein to NHS-5,6 carboxyfluorescein was mixed and incubated at room temperature for an hour in the dark. The mixture was then dialysed to remove the unconjugated carboxyfluorescein. The conjugated protein was stored at 4°C until further use. To achieve uniform liposome size, the mixture was extruded through a 200 nm polycarbonate membrane using an Avanti Polar Lipid Extruder at 45°C. The liposomal mixtures were visualised under transmission electron microscopy (TEM), whereas the size and zeta potential of the liposomal mixtures were determined by light scattering using a Malvern Zetasizer, whereby samples were diluted (1: 10) before measuring zeta potential and sizes. The concentration of the encapsulated protein was determined using a spectrofluorometer. The stability of the liposomes at refrigeration temperature was deduced from the changes in sizes of the liposomes using a Malvern Zetasizer. The morphology of liposomes was characterised using transmission electron microscopy (JOEL F200 TEM) with an acceleration voltage of 200 kV. A 10 pL sample was applied onto the TEM grid, followed by negative staining using 2% uranyl acetate.

[0157] Method - Optimisation of Liposomal Formulation

[0158] Different combinations of lipid components were experimented with to discern the suitability of the choice lipid components. To verify the responsiveness of the liposomes towards PLA2, dried lipid film was hydrated with fluorescein at a concentration above its quenching concentration and the release of the fluorescein with or without the presence of 80 pg / mL sPLA2 was read by spectrofluorometer at an excitation wavelength of 490 nm and emission wavelength of 514 nm and analysed. The quenching concentration of fluorescein was determined by reading the fluorescence intensity of the increasing amount of fluorescein. Quenching concentration is the concentration of fluorescein where the fluorescence intensity is levelled off after declining. The buffer solution used to dissolve the PLA2 was also pre-determined by comparing different buffer solutions (5 mM Tris buffer, 5 mM HEPES, 5 mM PBS buffer) and their effect on the release of fluorescein. The concentration of calcium ion (0-10 mM) was also trialled as the catalytic function of PLA2 is dependent on the calcium ion concentration. Dissolution of fluorescein can be carried out by first adding a volume of PBS solution to the fluorescein; sodium hydroxide (NaOH) was then added to the fluorescein solution drop-by-drop until the fluorescein was dissolved. The fluorescein solution was then filtered using Whatman™ 450nm filter paper under vacuum to remove any solid particles before the lipid hydration process.

[0159] Method - Release Rate

[0160] Investigation of the effect of incorporation of protein in liposomes on release rate is also of interest, especially for the determination of the functionality of protein-liposomes in the application of epicutaneous immunotherapy. The release of protein conjugated with NHS -fluorescein was read under a spectrofluorometer at an excitation wavelength of 494 nm and an emission wavelength of 519 nm. Conjugation of protein can be done by first mixing NHS -fluorescein with peanut protein in a 1:20 molar ratio for an hour at room temperature, the conjugated protein mixture was dialysed against PBS for two overnights. Protein concentration can be determined using BCA assay and spectrometer at an absorbance wavelength of 280 nm. Solutions containing the liposome-encapsulated protein with or without 80 pg / mL sPLA2 were incubated at 37 °C, and the liposomes were then ultracentrifuged using an Amicon Ultra Centrifugal Filter 100 kDa at x3,000g for 5 min. The concentration of released protein was deduced using a spectrofluorometer. To ensure that centrifugation of liposomes does not cause the liposomes to aggregate and lyse the liposomes, after each centrifugation the size of the liposomes was obtained using a zeta sizer. The effect of different concentrations (0-80 pg / mL) of enzymes on the release of protein from liposomes was compared.

[0161] Method - SDS-PAGE and Western Blot of Released Protein

[0162] Released protein with and without sPLA at 37 °C within seven hours were mixed with 4x Laemmli Sample Buffer and 3mg / mL of dithiothreitol. The mixture was heated at 95°C for 5 min and centrifuged at xl0,000g for 5 min. The 7 pg samples were applied onto each slot of Bio-Rad Mini-PROTEAN® TGX™ gels and run at 150V for 40 min. The gel was fixed using fixing solution (10% (v / v) of methanol, 0.7% (v / v) acetic acid in water) for 1 hr and stained with staining solution (0.275 g / L of Coomassie brilliant blue R250) for overnight. The gel was then de-stained using a de-staining solution (40% (v / v) methanol, 10% (v / v) acetic acid in water) for 3hr. The gel was read under the BioRad ChemiDoc Imaging system. Another gel was used for immunoblotting by first washing gel using milli-Q water, and then pre-soaking the gel in 20% ethanol for 5min. By using the Bio-Rad Trans-Blot Turbo Transfer System, the gel was blotted onto 0.2 pm of nitrocellulose membrane according to the manufacturer’s instructions. The blotted membrane was then washed and blocked with 1% BSA PBS blocking solution for 2 hr and then incubated with three separate rabbit anti- allergens (Ara hl, Ara h2 / h6 and Ara h3)-rabbit polyclonal IgG overnight. The band can be detected by incubating it with horseradish-peroxidase labelled goat anti-rabbit secondary antibody. SuperSignal West Dura substrate was used for the detection. The luminescence was captured using the ChemiDoc Imaging system. Each incubation was followed with three times washing using washing buffer (1% Tween20 in PBS).

[0163] Initial Formulation Studies

[0164] Changes in liposome size can be indicative of compromised stability, suggesting possible fusion or aggregation over time. As shown in Figure 1A-B, an equal ratio of DPPC / DOPE (1: 1, v / v) was initially selected due to it exhibiting good stability (i.e., low size increase (6%) but not significant) after seven days at 4 °C, along with higher encapsulation efficiency. The inclusion of stigmasterol was found to enhance liposome stability, as the size increase remained statistically insignificant (95% CI, T-test). As a result, an equal ratio of DPPC / DPPG was used for further optimisation of sPLA- responsive liposomes.

[0165] Another lipid component, DSPE-PEG, was also considered (Figure 1C). However, inclusion of DSPE-PEG in the liposome formulation was found to increase the zeta potential from -35.5 ± 3.0 mV to -3.2 ± 0.3 mV. DPPG / DPPC / stigmasterol was selected for further evaluation. It is believed that the strong negative charge of the selected formulation may enhance liposome stability by promoting repulsion between liposomes.

[0166] The percentage and type of sterol may also influence the size and polydispersity index (PDI) of liposomes. A lower PDI indicates greater homogeneity and improved stability of the liposomes. As shown in Table 1, 30% stigmasterol produced a lower PDI. Therefore, 30% stigmasterol was chosen over other formulations, including 30% cholesterol, 20% stigmasterol, and 10% stigmasterol.

[0167] Table 1: Particle characterisation of different weight percentage of sterol and DDPG, DP PC in a total lipid concentration of 2.5 mg / mL

[0168] The formulation taken forward for further experimentation was DPPG / DPPC / stigmasterol with a weight percentage of 35% / 35% / 30%.

[0169] Example 1.1 Thermoresponsive liposomes

[0170] Liposomes were characterized by their sizes, surface charge, encapsulation efficiency, stability and release rate. DLS was used to measure the average diameter size of the liposomes and zeta potential, the surface charge, of the liposomes.

[0171] Sizes and zeta potential of different lipid compositions in Table 2 were correlated.

[0172] Table 2 - Characterisation of different liposomal formulations

[0173] Stig. = stigmasterol, chol. = cholesterol.

[0174] The size and shape of liposomes were visualised using negative staining TEM. Liposomes can be observed to have a generally spherical morphology and their size is in nano-dimension in Figure ID (left). Negative-staining liposomes can cause aggregation of liposomes, however, the shape of the liposomes even after one month was retained, e.g. as seen in Figure ID (right), suggesting that the liposomes remains generally the same shape even after one month.

[0175] To further decrease the sizes of liposomes in order to investigate any potential increase of cellular uptake and permeability through the skin layers, liposomes were passed through a 200 nm PC membrane 21 times and through a 100 nm membrane another 21 times to achieve a smaller size. The extrusion method converts liposomes from multilamellar vesicles to large unilamellar vesicles, which facilitates the diffusion of liposomes through membranes. The sizes of the liposomes were analysed using DLS, which resulted in sizes of 128.5 ± 0.6 nm and 154.6+1.7 nm when extruded through lOOnm and 200nm respectively.

[0176] Encapsulation Efficiency

[0177] Crude peanut protein (PN) was encapsulated into 100 nm liposomes and 200 nm liposomes. A decrease in encapsulation efficiency (EE) from 21.6+2.2% to 15.8+2.9% was observed when passing through a 100 nm membrane after passing through a 200 nm. Accordingly, extrusion through 200 nm only was performed for the rest of the example. Furthermore, the difference between EE at 100 nm and 200 nm was significant using T- test (P<0.05). The optimisation of EE was performed by changing the buffer solution of the lipid component’s hydration media. PN required a buffer at a higher pH (pH 8.5) to ensure good protein solubility and extraction yield, especially for Ara h 2, one of the most potent peanut allergens. However, this pH buffer might influence the EE. It was found that the hydration buffer at pH 7.4 showed a higher encapsulation efficiency than the buffer solution at pH 8.4, therefore PBS at pH 7.4 was used as the hydration medium for the encapsulation of protein. PBS buffer was selected over Tris-HCl buffer because liposomes in PBS media demonstrated a narrower size distribution.

[0178] To calculate EE using Bradford Assay, concentration which embodies the encapsulated protein is calculated using a standard curve and subtracted with the concentration which represents empty liposomes. This reduction step is crucial as it appears that there is a reaction between Bradford assay with lipid components in liposomes. EE of PN protein is 21.6 + 2.2% when using Bradford Assay. With the use of a spectrofluorometer to measure the encapsulated NHS-carboxyfluorescein conjugated protein, EE of PN was found to be 11.7+0.7%. The significant difference between the measurement using Bradford Assay and using a spectrofluorometer could be attributed to the larger size of the protein after conjugation.

[0179] In this example, OVA, BSA, and Lf are chosen due to the difference in isoelectric point (pl), sizes (kDa) and the hydrophobicity of the purified protein. The sizes of OVA, BSA and Lf are 45kDa, 66.5 kDa and 80 kDa respectively, while the pl OVA, BSA and Lf are 5.2, 4.85 and 9.45 respectively. Interestingly, the EE of these purified allergens statistically does not differ significantly from each other using the T-test (P<0.05), no correlation of pl to EE and correlation of sizes to EE are found. It is noted that there is no correlation between the hydropathicity of the protein and EE, however, BSA is statistically higher than OVA. There were no significant differences in EE between the formulation of stigmasterol and the formulation of cholesterol as demonstrated in Figure 2B.

[0180] Stability studies

[0181] Stability studies in this study explored liposomes’ size distribution and liposomes’ integrity (Figure 2C). The increase in the size of liposomes was measured as an indicator of the presence of aggregation of liposome fragments, which represents the stability of the liposomes, using dynamic light scattering (DLS) Zetasizer. Different lipid components like DOPC, DMPC and DPPC were being compared. It has shown a significant difference in increment of sizes from 164.5+1.1 nm to 170.6+3.1 nm (T-test, P value<0.05) for DOPC / DOPE / stigmasterol stored at refrigeration temperature (4°C) after seven days. An increment of sizes from 164.2+2.4 nm to 200.3+1.0 nm can also be observed for DMPC / DOPE / stigmasterol (T-test, P value<0.001) after one month of storing at 4°C, while the size DPPC / DOPE / stigmasterol remain the same after one month in Figure 2C at 4°C and at 20°C. This indicates the stability of DPPC / DOPE / stigmasterol at refrigeration temperature and at room temperature.

[0182] Cumulative Release Rate

[0183] The in-vitro release behaviour can be studied using fluorescein-conjugated protein and centrifugal ultrafiltration method.

[0184] In Figure 3A, the release rate of quenched fluorescein from liposomes at 37°C showed a first-order release rate, whereby the release of content is rather fast in the beginning but slows down after some time. DPPC was utilised due to its high phase transition temperature. After 15 hours, 81.8% of the content in liposomes with formulations of DPPC was released while the content in liposomes with formulations of DOPC and DMPC released almost all its content (91.6% and 95.7% respectively) in the first 8 hours. DPPC, which has a phase transition temperature of 41 °C as compared to DOPC and DMPC which have a lower phase transition temperature of -16.5°C and 24°C, have a much lower release rate when compared to DOPC and DMPC.

[0185] To inquire about the differences in the release rate of PN and the release rate of just quenched fluorescein without PN, the release rate of PN experiment was conducted by first conjugating PN to NHS -fluorescein. The release rate of PN is rather different from the release rate of fluorescein, whereby the release rate of PN protein (in days) is slower than the release rate of fluorescein (in hours) in Figure 3B. A similar trend to the release rate of fluorescein is observed, whereby liposome formulation with DPPC has a lower release rate compared to the liposomes with DOPC and DMPC. After two weeks, all PN is released for DOPC and DMPC, however, with DPPC, 87.6 ± 9.5 % of its content is released. DPPC / DOPE / stigmasterol in Figure 3C has demonstrated that this formulation is suitable for heat-responsive liposomes.

[0186] Effect of Liposomes on Protein Encapsulation

[0187] Dot blot immunoassay of encapsulated protein is a qualitative analysis to detect the presence of protein and its binding nature by depositing protein followed by primary and secondary antibodies onto a membrane. Protein is allowed to form noncovalent bonds with the nitrocellulose membrane, a primary antibody is added to the protein and a secondary antibody that is compatible with the primary antibody and conjugated with horseradish peroxidase (HRP) is added later. Protein that binds to the primary antibodies can be distinguished from protein that does not have binding by adding the chemiluminescent reagents. In Figure 4A, the PN protein released from liposomes showed a reaction with rabbit antibodies (IgG), indicating that there is no effect on protein structure after being released from liposomes. Using ImageJ, the density intensity of the reaction in the dot blot is analysed. Released protein on day 1 and day 3 have significantly lower intensity density than the released protein on day 7, suggesting that there is more protein released on day 7. This is critical for the recognition of PN by the dendritic cells to produce appropriate IgG antibodies. A clearer indication can be seen using electrophoresis, whereby protein-encapsulated liposomes (PN-Lip) showed the same bands as protein (PN) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), while empty liposomes, which act as a control, have no protein in it as shown in Figure 4B.

[0188] Lipid components in liposomes can interfere with SDS-PAGE, as a result, liposomes were first centrifuged and filtered to remove the lipid components in liposomes before loading the samples into the well of SDS-PAGE. With the accumulative temperature at 37°C for seven days, PN protein was denatured to a smaller size, evidenced by the reduction of Ara h 1 at 61kDa to a lower protein band after being heated for more than 3 days. Without centrifuge and heating, no reduction of protein band was observed in fully released PN protein from liposomes, indicating that all major allergens in PN protein can be encapsulated inside the liposomes.

[0189] Western blot in Figure 4B was used to identify the major allergens of the released peanut protein, namely Ara h 1 and Ara h 2 / 6 with an exception for Ara h 3. These allergens were identified in day 1, 3 and day 7 released protein. Any loss of protein below a desirable level may be addressed using known stabilisers (for example, trehalose or sucrose could be added during the hydration process of the liposomes to preserve the shape of the PN protein and reduce denaturing of protein while increasing EE), temperatures, or other conditions.

[0190] Cytotoxicity Test

[0191] A cell viability assay (CKK) was conducted, to observe changes in the biochemical activities of living cells. According to ISO 10993-5, cell viabilities which are above 80% are considered non-toxic. The lowest cell viability percentage was 89.4+14.9% across all tested concentrations (10-100pg / mL) of liposomes, thus indicating that DPPC / DOPE / stigmasterol does not show toxicity and is safe to use. Example 1.2 - sPLA responsive liposomes Characterisation

[0192] The size of the DPPG / DPPC / stigmasterol liposomes was found to be 164.8 ± 2.0 nm using dynamic light scattering (DLS) and 120.8 ± 56.5 nm using transmission electron microscopy (TEM) (Figure 5A). The sizes determined by TEM are smaller as this technique measures particles under a dried state. On the other hand, DPPC / DOPE / stigmasterol had a smaller size at 154.6 ± 1.7nm using DLS and 76.7 ± 11.6 nm using TEM. The zeta potential of the DPPG / DPPC / stigmasterol liposomes was also measured using the ZetaSizer and was found to be (-35.5 ± 3.0) mV, which is lower than neutral DPPC / DOPE / stigmasterol which has a zeta potential of (-3.21 ± 0.28) mV. High zeta potential decreases the chances of aggregation and fusion of liposomes. It is believed that a shorter chain length can result in a decreased release rate and a lower phase transition temperature which may reduce the stability of the liposomes. Therefore, DPPG / DPPC / stigmasterol was determined to be a preferred lipid combination for synthesising stable sPLA-responsive liposomes. There were no significant changes in sizes at 4 °C with a PDI of 0.058 after 30 days, indicating that there was no aggregation of liposomes. This suggests that this formulation is stable at refrigeration temperature for at least a month. It is believed that as DPPG is a negative liposome, it tends to repel other such liposomes, which causes less aggregation and thus less leakage of protein / allergen caused by fusion of liposomes. Furthermore, stability studies were also performed at 20 °C, 37 °C and 50 °C. However, with the increasing storage temperature (20 °C, 37 °C and 50 °C), the sizes of the DPPG / DPPC / stigmasterol were found to increase by 20.3%, 10.4% and 2.4% respectively. Thus, a preferred storage condition for DPPG / DPPC / stigmasterol was found to be at 4°C.

[0193] Table 3. Polydispersity Index (DPI of the DPPG / DPPC / stigmasterol at 4 °C, 20 °C, 37 °C and 50 °C

[0194] Note: 0-day DPPG / DPPC / stigmasterol has been used as a control to be compared with. **** P <0.001, ** P<0.01, * P< 0.05.

[0195] Bradford assay was used to perform the encapsulation efficiency study of DPPG / DPPC / stigmasterol formulation, and it was found to be (35.9 ± 0.07) %. It was higher than the encapsulation efficiency of the DPPC / DOPE / stigmasterol, which has an encapsulation efficiency of 20%. Release Rate

[0196] The quenching concentration of fluorescein was found to be 25 mg / mL (Figure 5C). This self-quenched fluorescein was encapsulated inside the liposomes. In principle, when some fluorescein is released from the liposomes, the fluorescence intensity can be registered and read using a spectrofluorometer. Phospholipase A2 (PLA2) can be categorized into five major groups, namely secreted PLA2 (sPLA2), cytosolic PLA2 (cPLA2), Ca2+independent PLA2 (iPLA2), platelet-activating factor acetylhydrolase (PAF-AH) and lysosomal PLA2. For further study porcine pancreatic sPLA2 was selected due to the higher similarity to human sPLA2.

[0197] Phospholipase was found to coagulate with PBS, therefore phospholipase was dissolved in either Tris buffer or HEPES buffer at pH 7.4. Different buffer solution was used to examine the suitability of the buffer solution in dissolving phospholipase. The buffer includes calcium chloride to improve results, as phospholipase activity relies on calcium ion concentration and is sensitive to even low levels. This is coherent with the data in Figure 6A, whereby phosphate buffer solution with no calcium ions showed the lowest release rate among the other Tris buffer solutions. The release rate of fluorescein was still significantly higher (50% release rate after 15 hours) than without sPLA2 (40% release rate after 15 hours). This indicates that the liposomal formulation is responsive towards sPLA2. It is believed that the ester bonds of the phospholipid are hydrolysed by the enzyme. The phospholipase buffer with a higher calcium ion (10 mM CaCh) was selected to dissolve sPLA.

[0198] The effect of the concentration of sPLA2 was also investigated and it was found that with an increasing amount of sPLA2, the release rate increases as demonstrated in Figure 6B. For instance, when the sPLA2 was increased from 0 to 30pg / mL, the release rate increased 4.3-fold. To explore the disparity in the enzymatic effect of Group IB sPLA2 between cholesterol and stigmasterol, DPPG / DPPC / chole sterol and DPPG / DPPC / stigmasterol formulations were compared (Figure 6C). Interestingly, the incorporation of stigmasterol causes a higher release rate than cholesterol. The study underscores that the release kinetics of sPLA-responsive liposomes can be influenced by the nature of the sterol utilized.

[0199] The release rate of fluorescein-conjugated peanut protein was also determined to check whether the incorporation of protein affects the release profile. In Figure 6D, below 60 pg / mL of sPLA2 showed no significant difference in release rate (2% increase) when compared to no sPLA using T-test (P value =0.0634, confidence level, CI=95%). 80 pg / mL of sPLA2 was used in the study. As the formulation is temperature sensitive, due to its phase transition temperature, the release rate without sPLA2 was 89.9% higher at 50 °C than at 4 °C in Figure 6F. The release of the fluorescein-conjugated peanut protein reached pseudo after 3 hours for the examples without sPLA2, whereas with sPLA2, it continually increased until it reached 100%. In Figure 6G, there was a significant increment of release rate from 45.0+0.6% to 77.5+3.3% when comparing with sPLA2 and without sPLA2 at 37 °C. There were no significant differences in release rate (-12.4% increase, P value=0.505, confidence level, CI=95%) between with sPLA2 and without sPLA2 at 20 °C, suggesting sPLA2 activity is dependent on temperature, and requires a temperature higher than 20 °C, for example 37°C. The regulation of protein release is an interesting feature of this liposomal formulation, which precisely targets the epidermal layer where sPLA2 and Langerhans cells reside. By providing a controlled release, the formulation reduces premature protein release outside the epidermis, thereby maximizing protein delivery to the skin and effectively stimulating an immune response.

[0200] Released Protein Profile

[0201] The stability of the released protein was investigated as the immunoreactivity and immunogenicity of the protein should be maintained for effective allergen immunotherapy. In this research, the immunogenicity of the released peanut protein from liposomal formulation was explored using a dot blot against the IgG rabbit antibody, whereby it demonstrated that the immunoreactivity of the released peanut protein was preserved with or without sPLA2. The dot blot demonstrated that the immunogenicity has been reduced when compared to the native protein in Figure 7 A. In the allergen dilution ratio of 1: 10 (v / v), the intensity density of released protein is lower than the intensity density of protein.

[0202] Through the SDS-PAGE of the released protein, western blotting using IgG rabbit against peanut and western blotting against IgE in Figure 7B showed that Ara h 1 and Ara h 2, the most prominent peanut allergens, are present. However, the structure of the released protein, especially Ara h 1 had seemed modulated and broken down into a smaller molecular weight from 60 kDa to around 30 kDa. IgE human serum mostly is pooled samples of patients allergic to peanuts, especially against Ara h 2, therefore, main bands of Ara h 2 can be observed when blotting against human serum.

[0203] Permeability Study of Liposomes

[0204] The ability of liposomes to permeate through the skin layers to the stratum spinosum, where the Langerhans cells are primarily situated, was investigated due to the relevance to epicutaneous immunotherapy. Franz diffusion cells were used to study the permeability of liposomes across cellulose-type membranes in Figure 7C. In comparison with protein without liposomes, the amount that permeates through the membrane is nearer 3%, whereas with liposomes the amount of protein that permeates through is 10% after eight hours. This 65.2% increment is significant when analysed using T-test (P<0.05).

[0205] Cytotoxicity

[0206] In Figure 8, the Caco-2 cell viability at liposome concentration ranged from 10 pg / mL to 200 pg / mL, demonstrating that this DPPG liposome formulation does not cause cell toxicity to the colon cells in oral immunotherapy. Caco-2 cells were selected because they are derived from colon carcinoma and are usually used as a model for intestinal epithelial barrier. Furthermore, the cell viability test of the mouse embryonic fibroblast (MEF) cell line has demonstrated that this formulation is non-toxic to the fibroblast cell line.

[0207] Example 2 - Dissolving Microneedle Encapsulating Liposomes

[0208] Method - Liposome Preparation and Characterisation

[0209] To prepare the PN-encapsulated liposomes, the lipid components were dissolved in chloroform and dried to obtain a thin layer of lipids before rehydrating them with PN protein in PBS at 45 °C. The liposomal formulation was passed through a 200 nm polycarbonate membrane (PC) in an Avanti Polar Extruder at 45 °C. Sephadex G-50 was utilised to separate the encapsulated liposomes from the unencapsulated ones. The size and zeta potential of the PN-encapsulated liposomes were determined using Dynamic Light Scattering (DLS) Zetasizer. The encapsulation efficiency of PN in liposomes was predetermined using a spectrofluorometer, by first conjugating the PN with NHS-5,6- carboxyfluorescein in a 1:20 molar ratio. The liposomes were concentrated using a 3 kDa centrifuge filter at xl0,000g for 20-25 minutes to obtain 100 pL from 1 mL. The concentrated liposomes were resuspended, and their size was measured using DLS to ensure the stability of the liposomes after centrifugation (an increase in size being an indication of the occurrence of aggregation).

[0210] Method - Microneedle Preparation

[0211] A 12% Gantrez AN-139 polymer solution (w / v aqueous) was mixed with the concentrated liposomal solution at volume ratios of 1:4, 1: 1, 4: 1, and 9: 1. Subsequently, 80 pL of the mixed solution was pipetted onto the 19x19 PDMS mould (height: 600 pm) and placed in a vacuum chamber for 10 minutes to allow the mixed solution to flow into the microneedle tip and fill the cavity. Any air bubbles were removed from the solution by pipetting out the remaining solution. These steps were repeated twice to ensure that all microneedle channels were filled with the mixed solution. Additionally, a 24% Gantrez AN- 139 polymer or PVA or PCL was added to the PDMS without any liposomes as the backing. The microneedles were allowed to dry at room temperature for a day before being removed from the mould. The geometry and surface morphology of microneedles were captured using bright field and fluorescence microscopy. The swelling behaviour after inserting into the mimic skin model (10% gelatine in 1: 1 v / v glycerol / water) or porcine skin was also analysed using bright field / fluorescence microscopy.

[0212] Method - Microneedle Characterisation

[0213] The geometry and surface morphology of microneedles were captured using bright field and fluorescence microscopy. The swelling behaviour after inserting into the mimic skin model (10% gelatine in 1: 1 v / v glycerol / water) was also analysed using bright field / fluorescence microscopy.

[0214] Method - Skin Penetration Efficiency

[0215] Parafilm were cut into five equal sizes (1.5 x 1.5cm) and appropriately labelled. The five pieces of Parafilm will then be arranged layer by layer in sequential order. A thumb force 30N, previously calibrated using a load cell, was applied to the layered Parafilm for one minute. The pierced Parafilm was analysed using bright field microscopy, and the number of holes created by the microneedles was counted and compared among microneedles with different polymer-to-drug ratios. Additionally, the bending and occurrence of fracture of the microneedle tips after insertion was compared.

[0216] Similarly, a texture analyser was used to compress the microneedles with a force of 10- 100N, as opposed to using manual thumb force, and the similarity or dissimilarity of the results was noted. These results were also compared with the results of the penetrability of pig ear skin. Hairless pig ear skin was treated with 20% ethanol and covered with aluminium foil to prevent dehydration. Microneedles were then pressed onto the pig skin for one to ten minutes before being studied under the stereo microscope, confocal microscope, and scanning electron microscope (SEM).

[0217] Method - Microneedle Encapsulation Efficiency and Delivery Efficiency

[0218] The encapsulation efficiency of the liposomes in the microneedles can be measured using a spectrofluorometer. Initially, a known amount of NHS-5,6-carboxyfluorescein- conjugated protein is encapsulated in the liposomes. Subsequently, the liposomes are incorporated into the microneedles and allowed to dry. The microneedle tips are then removed using a scalpel and re-dissolved in Milli-Q water containing Triton X-100 to lyse the liposomes. The resulting solution is measured using a spectrofluorometer. Similarly, liposomes of the same concentration and volume without microneedles are also lysed using Triton X-100. The encapsulation efficiency can be calculated using the formula below.

[0219] Similarly, delivery efficiency can be calculated via the microneedle tip before inserted into the skin mimic model subtracting the microneedle tip after inserted into the skin mimic model. The microneedle tips before and after were removed using a scalpel carefully.

[0220] Method - Franz Diffusion Release of Microneedle

[0221] The Franz diffusion cell was assembled, with both the donor chamber and receiver chamber containing PBS and being separated by a 3 kDa SnakeSkin™ Dialysis Membrane or porcine skin (1 mm and 2 mm), a type of cellulose membrane. The microneedle was pressed into the membrane using the same thumb force for one minute before assembling the Franz cell setup for release. Additionally, a Franz diffusion release test using a control without microneedles was performed for comparison purposes. Samples of 200 pF each were taken from the receiver chamber of the Franz diffusion cells at specific time intervals: every 15 minutes for the first two hours, every 30 minutes for the following two hours, and then every hour for the remaining four hours. The receiver chamber was maintained at 37 °C and stirred at 150 rpm. An equal volume of fresh PBS was carefully added to the receiver chamber to replace the withdrawn samples, ensuring no air bubbles were introduced. Experiments were conducted in triplicate concurrently and repeated twice for accuracy. Method - Permeability of Released Protein

[0222] Agar was used to mimic skin function. After being punctured by the microneedles, the permeability of the fluorescently labelled protein was observed under the fluorescence microscope. Pig skin was also used to investigate the permeability of the fluorescently labelled protein by using a confocal microscope.

[0223] Method - Stability of liposomes after encapsulation

[0224] Microneedles were kept in a sealed aluminium bag containing nitrogen gas and were placed at room temperature and 4 °C for 0 days, a week and a month. Microneedle tips were removed using a scalpel carefully and redissolved in Milli-Q water (1 mL / patch). The solution was passed through a 0.45 pm PVDF membrane syringe fdter twice to separate the polymer from the liposomal solution. The sizes of the liposomes, whereby an increase in size is a good indication of instabilities caused by aggregation, were measured using a DLS ZetaSizer. The tip of the microneedle was also observed under a bright-field microscope to ensure that there is no hygroscopic effect on the microneedles which will then affect the penetrability of the microneedles. Using JOEL F200 transmission electron microscope (TEM), the released liposomes from the rehydrated microneedle were analysed structurally to check the integrity of the liposomes after storage for one week and one month. The released liposomes were placed on a carbon grid and negative stained using 2% uranyl acetate for 20 s before washing it. Stained liposomes were allowed to be dried for two overnights before reading using TEM.

[0225] Method - SDS-PAGE, Western blot and Dot Blot of Released Protein

[0226] In the same way, microneedle tips were removed using a scalpel carefully and redissolved in Milli-Q water (1 mL / patch) containing 10% triton-X-100. The solution was passed through a 0.45 pm PVDF membrane syringe fdter twice to separate the polymer from the liposomal solution. The solution was centrifuged further using a 100 kDa Amicon centrifuge filter at x3 ,000g for 20 minutes to separate the lipid components, before concentrating it with a 3 kDa Amicon centrifuge filter at x 10,000g for 20 minutes. The protein samples were heated at 95 °C with 4x Laemlli buffer and 3 mg / mL of dithiothreitol (DTT) for 5min. 20 pg of protein samples were placed in each well of the Mini-PROTEAN® TGX™ precast gel and run at 150V for 40min. The gel was washed with Milli-Q water before fixing it with a fixing solution for an hour. The gel was then placed in a staining solution overnight before de- staining it in a destaining solution for three hours.

[0227] Dot blot, which is a simple technique to detect protein using animal IgG antibodies and human IgE antibodies, was used to ensure that the protein structure is still recognizable by antibodies. 2 pL of protein samples were dotted onto a 0.45pm nitrocellulose membrane and allowed to dry for an hour. The membrane was blocked using a blocking buffer containing 1% BSA for an hour, before incubating the membrane in 1:5,000 primary rabbit IgG antibodies or 1:20 human serum at room temperature overnight. The membrane was then transferred into a 1:250,000 secondary goat antirabbit antibody (Abeam) or 1:50,000 secondary mouse antihuman antibody (Abeam) for an hour at room temperature. Each incubation was followed by the washing step using a washing buffer which contained 1% Tween 20 three times.

[0228] Western blot, which detects the specific protein that binds to the specific antibodies, was employed to determine which specific allergen was still present after the fabrication of the microneedle. These results were compared to the results of the native protein and protein loaded liposomes before the fabrication of the microneedle. Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) were run using Mini- PROTEAN® TGX™ precast gel. The gel was washed with Milli-Q water for five minutes to remove traces of sodium dodecyl sulfate which will affect the protein transfer. The gel was also treated with 20% ethanol on a rocking shaker for five minutes to increase the transfer efficiency. By using a Trans-Blot® Turbo™ Transfer System, the protein gel was transferred to a prewetted 0.2 pm nitrocellulose membrane (2.5A, 25V, 3min). Once the protein had been transferred to the nitrocellulose membrane, the membrane was washed using Milli-Q water and washing buffer. The membrane was then blocked for an hour at room temperature and incubated in primary antibodies overnight before incubating it in horseradish peroxidase (HRP)- conjugated secondary antibodies for an hour at room temperature. The membrane was read using BioradChemiDoc Imaging Systems after being treated with SuperSignal™ West Dura Extended Duration Substrate for five minutes.

[0229] Method - Fluorescence Intensity and Microfluidic Modulation Spectroscopy (MMS) The conformational changes of a protein can be determined using a spectrofluorometer to observe any changes in fluorescence intensity and shift in wavelength. Protein concentration was first determined via Bradford Assay. 200 pL of 1 mg / mL of each protein sample was added to a 96-well clear bottom black plate. Tryptophan (Trp), an amino acid present in peanut protein, has an absorbance at a wavelength of 280 nm and an emission wavelength of 300 nm-400 nm. Thus, the fluorescence intensity of each protein was measured using a spectrofluorometer. MMS was used to characterise the secondary structure of the protein. 700 pL of 1 mg / mL of protein samples in PBS were prepared and added to a 96-well plate. The samples were sonicated for 30 minutes before being analysed by AQS3pro (RedShiftBio). Triplicates of the samples were collected from the 96-well plate at a modulation rate of 1Hz and 5 psi backing pressure. The Higher Order Structure (HOS) was then measured using Gaussian curve fitting.

[0230] Method - Rat Basophil Leukemia (RBL) Immunological Assay

[0231] RBL assays were used to investigate the allergic response towards the released protein from the release of liposomes and microneedles. The RBL procedure followed that disclosed in Huang, L. et al. ‘A rapid and sensitive assay based on particle analysis for cell degranulation detection in basophils and mast cells’ Pharmacol Res 111, 374-383 (2016), with some minor changes. Briefly, RBL-2H3 cells, which contained FcERI (Fc for IgE antibodies), were cultured using 20% FBS and 1% Pen-Strep in DMEM. RBL- 2H3 were seeded onto a 96-well plate at 2xlO6 / mL. After stabilisation overnight, the cells were washed twice with Tyrode’s buffer (lx) before adding 1 : 10 human serum for another overnight. The serum was then removed and cells were washed twice with Tyrode’s buffer before adding 100 pL of peanut protein released from microneedle and allergen at a concentration ranging from 100- 500 pg / mL in Tyrode’s buffer with 50% (v / v) deuterium oxide for an hour. 40 pL of supernatant was collected and added to an equal volume of 80 mM 4-nitrophenyl N-acetyl-P-d-glucosaminidase in 0.1M citrate buffer pH 4.5 for an hour at 37°C. Lastly, 100 pL of 0.2M glycine buffer pH 10.7 was added, and absorbance was measured at 405nm in a spectrofluorometer. To obtain just the peanut protein, acetone protein precipitation was conducted. Acetone was allowed to reach -20 °C before adding it to the protein samples at a volume ratio of 1:4 (protein: acetone, v / v). The mixture was mixed and incubated for 1 hour at - 20 °C to allow further precipitation of the protein. The samples were then centrifuged at x 10,000g for 10 minutes. Acetone was then removed, and the residue of acetone was allowed to evaporate at room temperature for 30 minutes. The dry precipitates were allowed to be redissolved in PBS and the concentration of the protein was measured using Bradford Assay.

[0232] The compatibility of liposomes to the polymer solution

[0233] As the pH of 12% Gantrez solution was measured to be about pH 2, the effect on structural changes and sizes of DPPG / DPPC liposomes, when hydrochloric acid (HC1) buffer at pH 2 was added, was briefly investigated to ensure the suitability of the liposomes in the polymer solution. The results are presented in Table 4.

[0234] Table 4. The investigation of the addition of HC1 buffer at pH 2 to DPPG / DPPC liposomes.

[0235] H€'i btii&r Size Size standard H)I PDIstaadard

[0236] 60% 2S2.5 i .9 0.190 0.033

[0237] An increase in the sizes of DPPG / DPPC liposomes and their PDI could be observed when more than 30% of HC1 buffer at pH 2 was present in the solution mixture. When the mixture constituted more than 50% of the HC1 buffer, the PDI was increased from 0.1 to 0.2, indicating that the liposomes were no longer uniformly distributed, and lipid aggregation occurred.

[0238] Microneedle Geometry and Surface Morphology

[0239] The exemplary mould of the microneedle has a height of 600 pm, diameter of 300 pm, circular shape of the base microneedle, and interspacing of 300 pm. The height of the microneedle is selected to be 600 pm as a preferred height to reduce the interaction of pain receptors, but long enough to penetrate through the stratum spinosum where the Langerhans cells are located. Spacing between microneedles was also selected based on the balance of effectiveness in the permeation of active ingredients through the skin layers and the effectiveness of the insertion of microneedles. A decrease of spacings resulted in higher loading capacity and hence higher permeability through the skin layers while increasing the spacings can avoid the nail-bed effect, increasing penetration efficiency. An inverse mould made of PDMS was made by pouring the PDMS mixture onto the master mould and peeling off after being dried. PDMS was used as an inverse mould because it is chemically inert, flexible, heat-resistant and cost-effective.

[0240] Gantrez-AN-139, an alternating copolymer of methyl vinyl ether and maleic anhydride, was selected as the primary microneedle fabrication material due to its biodegradability and biocompatibility. Poly methyl vinyl ether exhibits a flexible property, while polymaleic anhydride provides the strength for penetration of the skin, thus rendering these co-polymers a good candidate for microneedles as a whole. From the bright-field microscopy images taken before the insertion test and analysed using ImageJ imaging software, the microneedles comprising the liposomal formulation were determined to have an average height of 598.4 ± 7.6 pm, an average diameter of 284.7 ± 4.1 pm, interspacing of 302.7 ± 12.7 pm and a structure similar to the master mould.

[0241] The microneedle tip after insertion was also comparable to the microneedle tip before insertion, and no breakage of the microneedle was observed even with a high liposomal formulation to polymer ratio of 9: 1, which verifies the superior strength of 12% Gantrez as a fabrication material for the microneedle when 10-20 N of force is applied. Nonetheless, with an application of force at 100N, breakage of the microneedle can be observed. The channels created by the different liposomes to polymer ratio were analysed using bright-field microscopy and holes on parafilm were also captured and numbered.

[0242] In this preliminary investigation of the selection of the ratio of a liposomal formulation to 12% Gantrez polymer, it was observed that a ratio of 9: 1 punctured through parafilm using thumb force (measured to be 30N). With decreasing liposomes to polymer ratio, the number of holes observed in each layer and the number of punctured parafilm layers were increased, which means an increase in penetration depth in Figure 9C. Nonetheless, microneedles with a ratio of 9: 1 were selected for a higher loading capacity.

[0243] Penetration Force of Microneedle

[0244] Bending and fracture of the microneedle tip are common failures for microneedles. To investigate microneedle failures, the effect of different forces (10N, 20N, 30N, 40N and 100N) using a texture analyser on the microneedle tips was investigated and found that there was no significant reduction (T-test, P<0.05, CI=95%) of microneedle tips except when more than 20 N force was applied to the microneedle tips in Figure 10A. A reduction of 15.7+0.5% in length can be observed when 30 N force is applied. Nonetheless, it was only at 30 N and above that, more than 80% of microneedles penetrated the parafilm in Figure 10B. This reduction observed was due to the bending and buckling of microneedle tips and this in turn reduced the sharpness of the microneedle tip, which caused the reduction of penetrability of the microneedle. This length reduction might also decrease the amount of active ingredients being delivered. The broken tips can be dissolved by the sweat and flow into the microneedle channel, hence recovering the loss amount. Nonetheless, reducing the force applied onto the microneedle to the skin could cause a reduction in the penetrability of the microneedle. As the tensile strength of Parafilm is around 2.45 MPa, the force required to puncture parafilm is 7.7 pN per microneedle tip (2 pm radius tip) using P = F / A, whereby P is the pressure (Pa), F is the force (N) and A is the area (m2). However, due to the nail-bed effect, a higher force is required to puncture the Parafilm.

[0245] Noticeably, a higher force is required to overcome the stratum corneum and epidermal layer than parafilm, as the young modulus of the stratum corneum is 2 MPa and the young modulus of the epidermis is 4 MPa. The mean ultimate tensile strength of human skin is approximately 27 MPa, which means that even higher forces are needed to break the skin. In Figure 10C, a significant increase (penetration efficiency increase from 2.3% to 47.5%) of microchannel formed on the porcine skin by piercing of the microneedle can be observed when more than 60 N of force is applied, and 85+5% of the microchannel can be observed when 100 N force is applied. Porcine pig ear skin was selected due to the many similarities in thickness of the stratum corneum, the thickness of the viable epidermis and the structure of hair follicles to that of human appendages. The differences in results when parafilm or porcine skin could be due to the nonuniform topology of the skin and the skin has a higher tensile strength than the parafilm. Furthermore, the maximum human thumb force ranged between 100.6 N to 137.6 N. As such, 100 N force was identified as a preferred penetration force to puncture the skin.

[0246] Penetration Depth of Microneedle

[0247] The top view of the microneedles channel formed in parafilm, mimic skin model and porcine skin were captured in Figure 10D. Each parafdm has a thickness of 130 pm. Using a 100 N texture analyser, 90% of the microneedle tips have a penetration depth of 130 pm, while 1% of microneedles with a ratio of 9: 1 achieved a penetration depth of 390 pm. The average thickness of the stratum corneum of subjects from the 18-66 age group is 12 pm and the epidermis can range from 40-110 pm depending on the body area. Therefore, this penetration depth is sufficient to puncture through the stratum corneum layer and reach the stratum spinosum where Langerhans cells are. On the other hand, the penetration depth of 90% of the microneedle tips in the mimic skin model is 300 pm. The penetration depth of 90% of the microneedle tips in cross-sectioned porcine skin is measured to be 62 pm in Figure 10E using ImageJ software. Whereby, with a 25pm radius of punctured holes in Figure 10F using a scanning electron microscope (SEM), an estimated 100 pm microneedle shaft is punctured through the porcine skin. The structure of punctured holes was also studied using a z-stack confocal microscope in Figure 10G.

[0248] Homogeneity of Microneedle

[0249] The homogeneity of the microneedle can affect the repeatability of the mechanical properties of the microneedle and reproducibility of the concentration of allergen-loaded liposomes encapsulated inside the microneedle tip. The homogeneity of the fluorescent microneedle tip was observed using a fluorescence microscope. There is no significant difference in mean intensity density per area (ranging from 34.6 + 2.7 to 37.4 + 2.6) when investigated using ImageJ software. The fluorescently labelled protein was evenly distributed throughout all microneedle tips without any patches (see Figure 11). This homogeneous distribution of liposomal formulation / polymer could provide a uniform distribution of active ingredients across the skin and minimise the variability between microneedle patches, resulting in predictable and reliable delivery of consistent dosages of active ingredients.

[0250] Microneedle Encapsulation Efficiency, Loading Capacity and Delivery Efficiency Loading capacity is defined as the amount of protein present in the microneedle tip. Loading capacity can be calculated as concentration used multiplied by the volume of liposomal formulation that filled into the 19x19 microneedle tips, the volume of the cone, V = l / Sin^h, whereby r is the radius of the base of microneedle and the h is the height of the microneedle. Therefore, the theoretical loading capacity of the microneedle would be 5 pg, however, only 3.13± 0.25 pg may be provided in the microneedle tip (determined using a spectrofluorometer). Encapsulation efficiency is defined as the amount of protein incorporated in liposomes detected in the microneedle over the amount of protein incorporated in liposomes that were added to the microneedle. The encapsulation efficiency in the microneedle tip was found to be 69.7 ± 5.6%. The delivery efficiency is identified as the delivered amount of protein incorporated in liposomes over the total amount of protein detected in microneedles. The delivery efficiency of protein after a minute is 86.5% ± 13.5%. Thus, 2.7 pg of protein per microneedle patch is being delivered across the skin.

[0251] Stability of liposome after encapsulation in microneedle

[0252] After encapsulation, the viability of liposomes was studied. The stability of the liposomes can be scrutinized using the changes in the size of the liposomes, whereby any signs of an increase in size may signify the occurrence of dismantling of the bilayer structure which may lead to aggregation and thus reduce the stability of the liposomes. No significant increase (T-test, P <0.05, confidence level, CI=95%) in sizes after encapsulation in microneedles was observed, where the average size was 122.4 ± 1.4 nm (Table 5). Sizes of the liposomes may influence the release rate of hydrophobic active ingredients but not hydrophilic active ingredients. Since there is no significant difference in sizes and surface peanut allergen is hydrophilic at above pH 4, there are expected to be no changes in the release kinetics of allergen from the liposomes. Nonetheless, the polydispersity index (PDI), which measures the uniformity of the size of liposomes, increased from 0.1 to 0.2, indicating that there was a shift from a more homogeneous sample size to a more heterogeneous sample size. The sizes of the liposomes in microneedles after a day, a week and a month at 20 °C and at 4 °C were also compared to check the storage stability of liposomes in microneedles. There was a further increment in the sizes of the liposomes after being rehydrated from a dried microneedle state for 0 days, one week and one month. Liposomes in a dry state encouraged the fusion of liposomes, as the liposomal particles were closely arranged in this state. Although there were no significant increases in sizes (T-test, P<0.05), the PDI increased from 0.2 to 0.6. This high dispersity of the sizes of liposomes may indicate the development of aggregated liposomes over time. These were further investigated using transmission electron microscopy (TEM) and it was shown that most of the liposomes retained in a spherical shape with a mean size of 84 ± 9 nm for one- week-old microneedle stored 4 °C and 122 ± 85 nm for one-month old microneedle stored at 4°C.

[0253] Table 5 - The investigation of the stability of liposomes in microneedles at 0 days, one week and one month using dynamic light scattering.

[0254]

[0255] The leakage of the quenched fluorescein from liposomes after rehydration was measured using a spectrofluorometer and determined to be 7.4 ± 1.8 % after incorporating with Gantrez and around 10.1+2.7% after being dried. This 37% increment in leakage percentages after being dried was not significant using a one-way ANOVA test (P value=0.3025, CI=95%). Subsequent storage at one week at 20°C and 4°C led to a significant 73.4% and 80.5% increment of leakage percentage respectively. The results of percentages of released fluorescein showed no significant differences between the storage at 4°C and storage at 20°C after a week (T-test, P value =0.6030, CI=95%) and after a month (T-test, P value=0.3858, CI=95%). There were also no significant differences between the percentages of released fluorescein after one week and after one month at 4°C (T-test, P value = 0.2536, CI=95%). See Figure 12A and B.

[0256] Protein profile after encapsulation in microneedle

[0257] SDS-PAGE was also used to check the stability of the protein after being encapsulated in liposomes and subsequently in microneedles. Acetone was used to remove the lipid components from the samples, which will affect the SDS-PAGE. Here we see that all protein bands from the liposomal formation that is encapsulated in the microneedle are the same as the native protein in Figure 13a. Thus, this study has presented a fabrication of a microneedle that neither used UV light nor high temperature to maintain protein integrity. The stability of the protein was also verified using western blotting against the surface antibodies of rabbit IgG specific to peanut and human serum IgE. The results confirmed that the immunoreactivity of the major potent allergen Ara h 2 remains. In Figure 13b, dot blot against rabbit peanut specific IgG has demonstrated a decreased intensity after encapsulation in microneedle, which showed that structural changes and epitopes may be being masked after encapsulation. Dot blot against human IgE has also shown reduced intensity after encapsulation, indicating a reduced immunoreactivity towards IgE.

[0258] To investigate potential structural changes post-encapsulation, a spectrofluorometer was used to measure the fluorescence intensity of the same concentration (1 mg / mL) protein. Changes in fluorescence intensity indicate that the tertiary structure of the protein has changed. This measured the fluorescence of Trp, an amino acid present in the protein. There was a red shift in the maximum emission wavelength from 320 to 330nm and a 22% decrease in fluorescence intensity from 1800 to 1400 in Figure 13c. This means that the protein structure has been unfolded after encapsulation, exposing linear epitopes while hindering structural epitopes. Change in the hydrogen bonding in protein due to incorporation in microneedle has caused the unfolding of beta-sheet (increase -23%) and folding beta-turn (increase 23%) in Figure 13e. In Figure 13f, there are no changes of beta-sheet or alpha-helix, stating that lipid - protein interaction does not have much influence on the structural changes. This indicates that the structural changes of the protein are likely to be due to the acidic conditions when Gantrez polymer is incorporated.

[0259] This structural change may lead to desirable reduced allergenicity of the peanut allergen. To investigate the structural epitope changes, a competitive MacroArray was conducted whereby a major protein allergen is printed onto the array and allowed to compete with the samples for the binding of the IgE human antibodies. Ara h 1 and Ara h 3 are major allergens in peanuts, while Ara h 2 is the most potent allergen in peanuts. In Table 6, Ara h 2 from acetone -precipitated microneedle with allergen-loaded liposomes has interacted with the antibodies. Thus, there was no binding of the antibodies and Ara h 2 printed on the array. Antibodies bind less to Ara h 1 and Ara h 3 from acetone-precipitated microneedles with allergen-loaded liposomes. This indicates that the structural changes caused a hindrance to the epitope. On the other hand, Ara h 6 from acetone-precipitated microneedle with allergen-loaded liposomes has more binding with the IgE antibodies.

[0260] Table 6. Competitive MacroArray to investigate the changes to the structural epitope.

[0261] IgE (kU / L) Control - With With l »g / snL without Frsteia Prorssis (MN

[0262] Competitor precipitate from acetone)

[0263] Ara h 3 S.65 0.34 2.27

[0264] Ara h S 0.55 0.70 0.49

[0265] Torsi IgE aaS 717 6W

[0266] Degranulation assay of rat-basophil leukaemia (RBL-2H3) mast cells was used to study the cellular response to an encapsulated allergen, by measuring the release of mediators, P-hexosaminidase. Reduced degranulation may indicate a reduction in allergenicity. A decrease in immunoreactivity does not always correlate with a reduction in allergenicity. A patient can be sensitized to the protein, meaning it binds with IgE, without experiencing an allergic reaction. The observed liposomes and microneedles did not exhibit significant cytotoxicity to RBL-2H3 cells. Acetone was used to precipitate the protein, so that the degranulation of the protein is only affected by the protein. In Figure 14 A, protein from acetone -precipitated microneedle with allergen-loaded liposomes was shown to exhibit a reduced allergenicity. This is further supported by the observed decrease in immunoreactivity.

[0267] Franz Release Rate and In vitro Permeability Test

[0268] Franz diffusion cell was used to evaluate the in vitro permeability of protein at 37 °C, with a 3 kDa regenerated cellulose membrane (SnakeSkin™ Dialysis Tubing). The delivery of protein greatly increased when a microneedle was used as compared to liposomes with crude peanut and crude peanut only. It was observed that 48.0+8.3% of the crude peanut was released from the liposomes and microneedle in 3 hours. The release of crude peanut protein follows zero order release kinetics, in which the release rate is almost constant for the first 5 hours before slowly plateauing at an 86.7% release percentage.

[0269] In vitro permeability through porcine skin was also investigated at different thicknesses (Figure 14B). With a 1 mm layer of porcine skin, there is more permeation of active ingredients. However, only 34.5+14.7% of the active ingredients permeate through the layers after eight hours. Increasing the layer of porcine skin by 1 mm (to 2 mm) led to a reduction of the permeation percentage by half (decrease from 34.5% to 17.7% at the eighth hour). This localised diffusion may improve the safety profile by potentially avoiding systemic side effects while potentially maintaining efficacy for an extended period. This may provide a longer opportunity for the recognition of Langerhans cells. Furthermore, the penetration of the skin using microneedle was capable of breaching the epidermal layer, in which Langerhans cells can be found in 33- 48 cell layers from the surface of the skin.

[0270] Dissolution of Microneedle and Swelling Behaviour

[0271] The dissolution of microneedle tips was observed using a fluorescence microscope. Using ImageJ software, it was concluded that 20.7 + 2.2 % of the microneedle tips were left after a minute and 9.7 + 3.2 % of the microneedle tips were left after five minutes of insertion in the mimic skin model in Figure 15a. When compared to the images taken before and after insertion in pig skin for a minute in Figure 15b, 38.6 + 3.1 % of microneedle tips were left. A complete dissolution of microneedle tips can only be observed after 10 minutes. This may indicate that the dissolution of microneedles in pig skin is slower than in the mimic skin. Accordingly, it is preferred that the microneedle be inserted for at least 10 minutes for complete dissolution in human skin. Due to the high dissolution rate, the microneedle patch was kept in the dark with nitrogen flushing and after 6 months, the microneedle was observed under a bright-field microscope. The 6-month old microneedle was found to retain its microneedle length, measured at 598.7 pm in Figure 15c using ImageJ software.

Claims

CLAIMS:

1. A liposome comprising a lipid component comprising or consisting essentially of or consisting of: dipalmitoy Ipho sphocholine (DPPC ) ; dipalmitoylphosphatidylglycerol (DPPG); and a sterol.

2. The liposome of claim 1, wherein the sterol is stigmasterol.

3. The liposome of claim 1 or claim 2, wherein the DPPC is present in amount of about 30% by weight to about 40% by weight, about 32% by weight to about 38% by weight, or about 34% by weight to about 36% by weight, relative to the total weight of the lipid component.

4. The liposome of any one of claims 1 to 3, wherein the DPPG is present in amount of about 30% by weight to about 40% by weight, about 32% by weight to about 38% by weight, or about 34% by weight to about 36% by weight, relative to the total weight of the lipid component.

5. The liposome of any one of claims 1 to 4, wherein the sterol is present in amount of about 25% by weight to about 35% by weight, about 27% by weight to about 33% by weight, or about 29% by weight to about 31% by weight, relative to the total weight of the lipid component.

6. The liposome of any one of claims 1 to 5, wherein the DPPC is present in amount of about 30% by weight to about 40% by weight; wherein the DPPG is present in amount of about 30% by weight to about 40% by weight; and wherein the sterol is present in amount of about 25% by weight to about 35% by weight, relative to the total weight of the lipid component.

7. The liposome of any one of claims 1 to 6, which is anionic.

8. The liposome of any one of claims 1 to 7, further comprising a protein.

9. The liposome of claim 8, wherein the protein is an antigen.

10. The liposome of claim 9, wherein the antigen is a peanut-derived allergen.

11. The liposome of claim 9 or claim 10, wherein the antigen is selected from Ara h 1, Ara h 2, Ara h 3, Ara h 6 or a combination thereof.

12. The liposome of any one of claims 8 to 11, wherein the protein is encapsulated by the liposome.

13. The liposome of any one of claims 1 to 12, wherein the liposome further comprises one or more additional lipids selected from the group consisting of: neutral lipid, structural lipid and PEGylated lipid.

14. The liposome of any one of claims 1 to 13, wherein the liposome has a mean diameter (in nm) between about 120 and about 200, between about 140 and about 200, between about 130 and about 190, between about 140 and about 180, between about 140 and about 160, or between about 150 and about 170.

15. The liposome of any one of claims 1 to 14, wherein the liposome has a Zeta potential (mV) between about 0 and about -150, between about -10 and about -100, between about -20 and about -80.

16. The liposome of any one of claims 1 to 15, wherein the liposome has a Zeta potential (mV) between about -30 and about -40.

17. The liposome of any one of claims 1 to 16, wherein the liposome is stable for at least 30 days at a temperature of about 4 °C.

18. The liposome of any one of claims to 1 to 17, wherein the liposome has a release rate about or greater than about 40% after 10 hours in Tris buffer at a pH of 7.4 at 37 °C in the presence of sPLA2 at a concentration of 80 pg / mL and CaCh at a concentration of 10 mM.

19. A liposomal composition comprising the liposome of any one of claims 1 to 18, and a polymer.

20. The liposomal composition of claim 19, wherein the polymer is selected from the group consisting of polyvinyl alcohol, polyvinylpyrrolidone, polyethylene glycol, polylactic acid, polyglycolic acid, poly(lactic-co-glycolic acid), polycaprolactone, hyaluronic acid, chitosan, gelatin, carboxymethyl cellulose, hydroxypropyl methylcellulose, poly(y-glutamic acid), starch-based polymers, xanthan gum, alginate, dextran, pullulan, pectin, poly(2-hydroxyethyl methacrylate), polyvinyl acetate, polyaspartic acid, polyhydroxyethyl acrylate,polyhydroxybutyrate, polyhydroxyvalerate, polyethylene oxide, polypropylene fumarate, methylcellulose, hydroxyethyl starch, carrageenan, polymers of maleic anhydride, polymers of vinyl ether or methyl vinyl ether, and copolymers of monomers thereof.

21. The liposomal composition of claim 19 or claim 20, wherein the polymer is a copolymer of methyl vinyl ether and maleic anhydride, optionally an alternating copolymer of methyl vinyl ether and maleic anhydride.

22. The liposomal composition of any one of claims 19 to 21, wherein the copolymer of methyl vinyl ether and maleic anhydride is Gantrez AN- 139.

23. The liposomal composition of any one of claims 19 to 22, wherein the ratio of the concentration (w / v) of the polymer of the liposomal composition and the concentration (w / v) of the lipid components of the liposome in the liposomal composition, is between about 10: 1 and about 1: 10, between about 6: 1 and about 1:2, between about 6: 1 and about 1:1, between about 6: 1 and about 2: 1, between about 6: 1 and about 3: 1, between about 6: 1 and about 4: 1, or between about 6: 1 and about 5: 1.

24. The liposomal composition of any one of claims 19 to 23, wherein the concentration of the polymer is about or less than about 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, 3, or 1.5% (w / v).

25. The liposomal composition of any one of claims 19 to 24, wherein the concentration (in % w / v) of the lipid components of the liposome is between about 0.025 and about 1, between about 0.05 and about 0.4, or between about 0.2 and about 0.3.

26. A microneedle array comprising a plurality of microneedles comprising the liposome or liposomal composition of any one of claims 1 to 25.

27. The microneedle array of claim 26, wherein the plurality of microneedles are dissolvable.

28. The microneedle array of claim 26 or claim 27, wherein the plurality of microneedles has: a mean height (in pm) of between about 400 and about 1000, between about 500 and about 700, between about 550 and about 650, or between about 580 and about 620; and / ora mean base diameter (in pm) of between about 250 and about 350, or about 280 and about 300.

29. The microneedle array of any one of claims 26 to 28, wherein the microneedle array has an encapsulation efficiency (in %) greater than about 50, 55, 60 or 65.

30. The microneedle array of any one of claims 26 to 29, wherein the microneedle array has a delivery efficiency (in %) greater than about 50, 55, 60, 65, 70, 75, 80 or 85, after 1, 2, 4, 6 or 8 hours.

31. The microneedle array of any one of claims 26 to 29, wherein the microneedle array has a loading capacity of at least 3 pg.

32. The microneedle array of any one of claims 26 to 31, when read on claim 8, wherein the microneedle array is capable of delivering about or greater than about 1.0, 1.5, 2.0 or 2.5 pg of the protein to human skin.

33. A skin patch comprising the microneedle array of any one of claims 26 to 32.

34. A method of desensitising a subject to an allergen, the method comprising delivering: the liposome of any one of claims 8 to 18; or the liposomal composition of any one of claims 19 to 25, to the subject percutaneously or epicutaneously, wherein the liposome or liposomal composition comprises the allergen.

35. A method of delivering an antigen to a subject, the method comprising delivering: the liposome of any one of claims 8 to 18; or the liposomal composition of any one of claims 19 to 25, to the subject percutaneously or epicutaneously, wherein the liposome or liposomal composition comprises the antigen.

36. The method of claim 34 or claim 35, wherein the liposome or liposomal composition is delivered using: a microneedle array, optionally the microneedle array of any one of claims 26 to 32; and / or a skin patch, optionally the skin patch of claim 33.

37. Use of the liposome of any one of claims 8 to 18 or the liposomal composition of any one of claims 19 to 25, in the densensitising of a subject to an allergen, wherein the liposome or liposomal composition comprises the antigen.

38. Use of the liposome of any one of claims 8 to 18 or the liposomal composition of any one of claims 19 to 25, in the delivery of an antigen to a subject, wherein the liposome or liposomal composition comprises the antigen.

39. Use of the liposome of any one of claims 8 to 18 or the liposomal composition of any one of claims 19 to 25, in the manufacture of a medicament for the densensitising of a subject to an allergen, wherein the liposome or liposomal composition comprises the antigen.

40. Use of the liposome of any one of claims 8 to 18 or the liposomal composition of any one of claims 19 to 25, in manufacture of a medicament for the delivery of an antigen to a subject, wherein the liposome or liposomal composition comprises the antigen.

41. The use of any one of claims 37 to 40, wherein the liposome or liposomal composition is to be delivered or formulated to be delivered percutaneously or epicutaneously.

42. The liposome of any one of claims 8 to 18, or the liposomal composition of any one of claims 19 to 25, or the microneedle array of any one of claims 26 to 32, or the skin patch of claim 33, for use in desensitising a subject to an allergen, wherein the liposome or liposomal composition comprises the allergen.

43. The liposome of any one of claims 8 to 18, or the liposomal composition of any one of claims 19 to 25, or the microneedle array of any one of claims 26 to 32, or the skin patch of claim 33, for use in delivering an antigen to a subject, wherein the liposome or liposomal composition comprises the antigen.

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