[0001] This invention relates to composite materials for controlled release, for example, for use in drug delivery. The invention also provides methods for making the composite materials, formulations comprising the composite materials, and uses of said composite materials and formulations.

BACKGROUND

[0002] Transdermal drug delivery (TDD) has various advantages (ease of use, better patient compliance, improved bioavailability and fewer adverse effects, etc.) over other drug delivery options (oral and injections) and has gained increased attention over the last decade. TDD of biologies (e.g. peptides, proteins, nucleic acids) and the use of new drug delivery devices such as microneedle systems for TDD, have high growth potential and are likely to have more activity in coming years. Passive TDD systems do not disrupt the skin to facilitate drug absorption into systemic circulation. These systems rely on natural diffusion to transfer active pharmaceutical ingredients (APIs) to the skin and then to systemic circulation. The major limitation with passive TDD technology is its inability to deliver active substances with larger molecules like peptides, proteins and vaccines. Active TDDs disrupt the stratum corneum (the primary barrier of the skin) facilitating drug absorption into systemic circulation. These methods include chemical enhancers and permeators, mechanical aids, low electrical current, thermal ablation, and/or microneedles.

[0003] Known means of sustained drug delivery via active transdermal delivery systems are limited by quick drug delivery rates and durations, typically lasting for a period of only a few hours. Thus, these known systems are generally unsuitable for use in the treatment of chronic medical conditions that require long-term medications. Additionally, known systems often require harsh manufacturing conditions and the use of environmentally unsustainable materials.

[0004] Accordingly, there is a need in the art for a system which facilitates efficient, sustained drug delivery over longer periods of time. There is also a need to develop a system that is environmentally sustainable and involves facile manufacturing conditions.

BRIEF SUMMARY OF THE DISCLOSURE

[0005] In accordance with a first aspect of the present invention there is provided a composite material comprising: a first polymer; and a second polymer, wherein the first polymer is a cross-linked polymer capable of forming a porous hydrogel on exposure to water; wherein the second polymer is more soluble than the first polymer in water; wherein upon exposure to water, a system is formed in which the second polymer is situated in the pores of the porous hydrogel formed from the first polymer. Typically, the first polymer is in the form of a cross-linked porous network and the second polymer is situated in the pores of the porous network,

[0006] In accordance with a second aspect of the present invention there is provided a composite material comprising: a first polymer, wherein the first polymer comprises cross-linked chitosan; and a second polymer, wherein the second polymer is polyvinylpyrrolidone (PVP). Typically, the first polymer is in the form of a cross-linked porous network and the second polymer is situated in the pores of the porous network,

[0007] The composite material of the present invention is particularly useful in drug delivery applications. For example, the composite material may further comprise a therapeutically effective amount of a pharmaceutically active compound, or pharmaceutically acceptable salt thereof.

[0008] The composite material of the first or second aspect is also useful in agronomic applications. For example, the composite material may further comprise an effective amount of an agronomically active compound, or agronomically acceptable salt thereof.

[0009] The composite material achieves several advantages over prior art delivery platforms (e.g. drug delivery platforms).

[0010] The water may be comprised in a physiological environment. It may be that the first polymer is a cross-linked polymer capable of forming a porous hydrogel on exposure to a physiological environment; the second polymer is more soluble than the first polymer in the physiological environment; and that upon exposure to the physiological environment, a system is formed in which the second polymer is situated in the pores of the porous hydrogel formed from the first polymer.

[0011] The second polymer is more soluble in the physiological environment than the first polymer. It may be that both the first and second polymers are soluble in the physiological environment, the first polymer being less soluble than the second polymer. Alternatively, it may be that the first polymer is insoluble in the physiological environment, while the second polymer is soluble in the physiological environment. It may be that the first polymer is insoluble in the physiological environment but is biodegradable in the physiological environment. It may be that the first polymer is insoluble and biodegrades in the physiological environment more slowly than the second polymer dissolves in the physiological environment. For example, the first polymer may biodegrade in the physiological environment over a period of at least 4 weeks. The use of two such polymers having differing solubilities in the composite material of the invention means that the mechanical properties of the composite material are highly tunable. The second polymer may be selected to provide particular mechanical properties to the composite that are useful for implantation of a device made of the composite. For example, the second polymer may be selected to provide a soft material (e.g. for an implantable hydrogel), a hard material (e.g. for microneedles), or a material capable of transitioning between the soft and hard states. The first polymer may be selected to provide properties (either mechanical or in terms of drug release) that are desired once the device has been implanted and the second polymer has dissolved.

[0012] When the composite material further includes an active agent, the solubility of the first and second polymers in a physiological environment enables the tuning of the release profile of the active agent. In particular, when the composite material of the invention is placed in water, the first polymer will form a porous hydrogel and the second polymer will dissolve, causing the first polymer to release the active agent. When the active agent is a pharmaceutically active compound, controlling the relative solubility of the first and second polymers allows greater control of the drug delivery profile.

[0013] Typically, the active agent (e.g. pharmaceutically active compound) might be comprised in the first polymer. When the composite is exposed to water (e.g. in a physiological environment), the second polymer rapidly dissolves, leaving the porous first polymer which can slowly release the active agent (e.g. pharmaceutically active compound). This can result in continuous and sustained release of the active agent (e.g. pharmaceutically active compound) over a longer period of time (e.g. days to months) from the hydrogel resulting from the first polymer. In addition, the active agent (e.g. pharmaceutically active compound) or a different active agent may be comprised in the second polymer. Thus, the composite can provide an initial burst of the active agent (e.g. pharmaceutically active compound) or different active agent (e.g. different pharmaceutically active compound) in addition to the sustained release. Thus, when the active agent is a pharmaceutically active compound, the composite material of the invention provides greater control of drug delivery rate and duration compared to prior art formulations.

[0014] In a third aspect of the invention, there is provided a method of making a composite material, the method comprising: providing a mixture comprising a first polymer and a second polymer in a solvent, gelating the mixture to form a gelated mixture, and drying the gelated mixture to form the composite material. The composite material may be a composite material of the first or second aspects.

[0015] In a fourth aspect of the invention there is provided a composite material obtainable or obtained by the method of third aspect.

[0016] In a fifth aspect of the invention there is provided a transdermal patch comprising a composite material of the first, second or fourth aspect, the transdermal patch comprising a body and a plurality of microneedles attached to the body, wherein the plurality of microneedles comprises the composite material of the first aspect or third aspect.

[0017] Known transdermal patches comprising microneedles deliver a pharmaceutically active compound over short periods of time (i.e. several minutes to hours). In contrast, the transdermal patch of the invention may be tuned to provide quick delivery of a pharmaceutically active compound over a short period of time (i.e. several minutes to hours) and/or continuous and sustained release over longer periods of time (i.e. days to months) by manipulating the relative solubilities of the first and second polymers of the composite material present in the patch in the same manner as discussed above in relation to the first aspect. Additionally, the body attached to the microneedles may acts as a reservoir of pharmaceutically active compound that delivers the pharmaceutically active compound to the patient via the microneedles. The rate at which the pharmaceutically active compound is delivered to the patient may be controlled by manipulating the mechanical properties of the microneedles, the porosity of the composite (arising from the structure formed by the first polymer), the solubility of the microneedles, and the mechanical properties of the body.

[0018] The transdermal patch of the fifth aspect also includes all of the advantages of the composite material of the first aspect, the second aspect and the fourth aspect.

[0019] In a sixth aspect of the invention there is provided an implantable device comprising a composite material of the first, second or fourth aspect.

[0020] The implantable device of the sixth aspect includes all of the advantages of the composite material of the first aspect, the second aspect and the fourth aspect.

[0021] The composite material of the first aspect, second aspect or fourth aspect may be bioresorbable in vivo. Thus, the implantable device of the sixth aspect has the advantage of not needing to be retrieved from a patient following treatment, thereby reducing the amount of surgical intervention required.

[0022] In a seventh aspect of the invention there is provided a transdermal patch of the fifth aspect or an implantable device of the sixth aspect for use as a medicament. [0023] In an eighth aspect there is provided a method of administering a pharmaceutical to a patient in need thereof, the method comprising applying a transdermal patch of the fifth aspect to the patient.

[0024] In a ninth aspect there is provided a method of administering a pharmaceutical to a patient in need thereof, the method comprising implanting the implantable device of the sixth aspect in the patient.

[0025] In a tenth aspect of the invention there is provided a use of the composite material of the first aspect, second aspect or fourth aspect to administer a pharmaceutically active compound, or pharmaceutically acceptable salt thereof, to a patient.

Composite Material

[0026] The first polymer may comprise one or more cross-linked polymers capable of forming a hydrogel. Thus, the first polymer may comprise a cross-linked polymer network, i.e. a cross-linked porous polymer network. The first polymer may comprise one or more cross-linked polymers selected from: cross-linked chitosan, cross-linked gelatin, crosslinked hyaluronan, cross-linked cellulosics, cross-linked polymethacrylates, cross-linked poloxamers, and mixtures thereof. Preferably, the first polymer comprises cross-linked chitosan.

[0027] The first polymer may be cross-linked by a crosslinking agent selected from: genipin, glutaraldehyde, sodium tripolyphosphate, and hexamethylenediamine. Preferably, the first polymer is cross-linked by genipin.

[0028] The cross-linking agent may be present in an amount of from about 0.001 % to about 3.5% by weight of the first polymer. The cross-linking agent may be present in an amount of from about 0.01% to about 0.5% by weight of the first polymer. The cross-linking agent may be present in an amount of from about 0.2% to about 0.35% by weight of the first polymer.

[0029] The cross-linking in the first polymer may be covalent cross-linking, non-covalent cross-linking, or a mixture of covalent and non-covalent cross-linking.

[0030] The first polymer may be present in an amount of no more than about 40%, no more than about 30%, no more than about 20% or no more than about 10% by weight of the composite material.

[0031] The first polymer may be present in an amount of from about 1 % to about 90%, from about 1% to about 60%, from about 1% to about 40%, from about 1% to about 25%, from about 1% to about 15% by weight of the composite material. The first polymer may be present in an amount of from about 1% to about 90%, from about 2% to about 60%, from about 5% to about 40%, from about 5% to about 25%, from about 5% to about 15% by weight of the composite material. The first polymer may be present in an amount of from about 2% to about 8% by weight of the composite material.

[0032] The first polymer may be cross-linked chitosan present in an amount of no more than about 40% by weight of the composite material. The cross-linked chitosan may be present in an amount of less than about 10% by weight of the composite material.

[0033] The hydrogel formed from the first polymer is porous. For example, the hydrogel formed from the first polymer may be in the form of a polymeric network having pores. The second polymer is situated within the pores of the porous hydrogel formed from the first polymer. The second polymer may interact with the hydrogel formed from the first polymer via non-covalent interactions, including ionic interactions and hydrogen bonding.

[0034] The porosity of the first polymer (i.e. the proportion of the first polymer by volume which is not solid) may be from about 10% to about 99%. The porosity of the first polymer may be from about 30% to about 75%. The porosity of the first polymer may be from about 40% to about 65%.

[0035] The second polymer may be present in an amount of at least 10% by weight of the composite material. The second polymer may be present in an amount of at least 25% by weight of the composite material. The second polymer may be present in an amount of at least 50% by weight of the composite material. The second polymer may be present in an amount of at least 60% by weight of the composite material. The second polymer may be present in an amount of at least 75% by weight of the composite material. The second polymer may be present in an amount of at least 90% by weight of the composite material. The second polymer may be present in an amount of at least 95% by weight of the composite material.

[0036] The second polymer may be present in an amount of from about 15% to an amount of about 95% by weight of the composite material. The second polymer may be present in an amount of from about 15% to an amount of about 90% by weight of the composite material. The second polymer may be present in an amount of from about 15% to an amount of about 75% by weight of the composite material. The second polymer may be present in an amount of from about 15% to an amount of about 50% by weight of the composite material. The second polymer may be present in an amount of from about 15% to an amount of about 25% by weight of the composite material. These lower amounts of the second polymer are particularly useful where the active agent is a biopolymeric drug.

[0037] The second polymer may be present in an amount of from about 25% to an amount of about 95% by weight of the composite material. The second polymer may be present in an amount of from about 50% to an amount of about 95% by weight of the composite material. The second polymer may be present in an amount of from about 60% to an amount of about 95% by weight of the composite material. The second polymer may be present in an amount of from about 75% to an amount of about 95% by weight of the composite material. The second polymer may be present in an amount of from about 85% to an amount of about 95% by weight of the composite material. The second polymer may be present in an amount of from about 90% to an amount of about 95% by weight of the composite material. These higher amounts of the second polymer are particularly useful where the active agent is a small molecule.

[0038] Without wishing to be bound by theory, it is thought that the second polymer being present in an amount of at least 60% (e.g. at least about 75%) by weight of the composite results a higher percentage of the pore volume in the first polymer porous network being occupied by the second polymer, resulting in increased mechanical strength of the composite material. The second polymer may fill the pores of the network formed by the first polymer. For example, the second polymer may occupy at least 90% (optionally at least 95%) of the pores of the porous hydrogel formed from the first polymer. Where the amount of the second polymer is high, the composite material may be suitably strong that it is capable of penetrating the skin of a patient without breakage of the composite material.

[0039] The second polymer may have an average molecular weight of from about 2,000 gmol' ¹ to about 1 ,400,000 gmol' ¹ , from about 30,000 gmol' ¹ to about 90,000 gmol' ¹ , from about 40,000 gmol ^-1 to about 70,000 gmol' ¹ , or from about 50,000 gmol' ¹ to about 60,000 gmol' ¹ . It may be that the second polymer has an average molecular weight of from about 55,000 gmol' ¹ to about 60,000 gmol' ¹ .

[0040] The second polymer may be selected from: polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), polyethylene glycol (PEG), cellulosics (e.g. hydroxypropyl cellulose, hydroxyethyl cellulose, carboxymethylcellulose). Preferably, the second polymer is polyvinylpyrrolidone (PVP).

[0041] The second polymer may be a vinyl polymer. For example, the second polymer may be polyvinylpyrrolidone (PVP) or polyvinyl alcohol (PVA).

[0042] The second polymer may be linear. Alternatively, the second polymer may be partially or fully branched.

[0043] The second polymer may not be cross-linked. [0044] The second polymer may be present in an amount of from about 99% to about 10%, from about 90% to about 95%, or from about 92% to about 94% by weight of the composite material.

[0045] The second polymer may be polyvinylpyrrolidone (PVP) present in an amount of at least 60% by weight of the composite material. The second polymer may be polyvinylpyrrolidone (PVP) present in an amount of at least 75% by weight of the composite material. The polyvinylpyrrolidone (PVP) may be present in an amount of at least 90% by weight of the composite material.

[0046] The composite material may be biocompatible, i.e. it may comprise biocompatible first and second polymers. It may be that the first polymer is biocompatible. It may be that the second polymer is biocompatible.

[0047] The composite material may further comprise an active agent. The active agent may be a pharmaceutically active compound or an agronomically active compound.

[0048] The active agent may be a pharmaceutically active compound, or pharmaceutically acceptable salt thereof, in a therapeutically effective amount. The pharmaceutically active compound may be a small molecule, e.g. it may have a molecular weight below 1000 g/mol. The pharmaceutically active compound may have a molecular weight below 500 g/mol. Alternatively, the pharmaceutically active compound may be a biopolymeric drug, e.g. a protein, an antibody or conjugate thereof, a polysaccharide, a polynucleotide. Exemplary drugs that might be released include those that are used to treat a disease selected from pain, arthritis, diabetes, asthma, cardiovascular disorders, cancer, Alzheimer’s disease, Parkinson’s disease and other neurological conditions.

[0049] The active agent may be an agronomically active compound, or agronomically acceptable salt thereof, in an agronomically effective amount. The agronomically active compound may be a fertiliser, a fungicide, a pesticide or an insecticide.

[0050] It may be that the active agent is admixed with the second polymer.

[0051] When the active agent is admixed with the second polymer, it may be that lower amounts of the second polymer are present. For example, when the active agent is present, the second polymer may be present in an amount of less than about 90%, less than about 70%, less than about 50%, less than about 30% or less than about 25% by weight of the composite material. When the active agent is present, the second polymer may be present in an amount of less than about 40%, less than about 30% or less than about 25% by weight of the composite material. The second polymer may be present in an amount of at least 16% by weight of the composite material. In these embodiments, the active agent is typically a biopolymeric drug, e.g. a protein, an antibody or conjugate thereof, a polysaccharide, a polynucleotide.

[0052] The composite material may further comprise an organic compound comprising an alkyne, and a transition metal catalyst comprising palladium, platinum, iron, copper, cobalt, ruthenium, rhodium or iridium. The transition metal catalyst may be suitable for catalysing an oscillatory oxidative carbonylation reaction of the organic compound. The transition metal catalyst may comprise palladium. The transition metal catalyst may comprise Pd ²⁺ , e.g. Pdh or PdCh. The transition metal catalyst may comprise cobalt. The transition metal catalyst may comprise cobalt ² *, e.g. Co(NOs)2.6H2O.

[0053] The transition metal catalyst may be bound to the first polymer.

[0054] The composite material may further comprise a biopolymeric substrate. The biopolymeric substrate may be a PEG-based polymer. The biopolymeric substrate may be admixed with the second polymer.

[0055] Upon exposure to water, the first polymer forms a hydrogel. When present, the transition metal catalyst may be distributed homogeneously through the hydrogel. Alternatively, the hydrogel may comprise areas of higher concentration of the transition metal catalyst and areas of lower concentration of the transition metal catalyst.

[0056] It may be that the organic compound is bound (e.g. covalently attached) to the first polymer.

[0057] Upon exposure to water, the first polymer forms a hydrogel. The organic compound may be distributed homogeneously through the hydrogel. Alternatively, the hydrogel may comprise areas of higher concentration of the organic compound and areas of lower concentration of the organic compound.

[0058] The catalyst may be suitable for catalysing a sustained oscillatory reaction of the organic compound in the hydrogel in response to a non-physical stimulus, causing the hydrogel to undergo oscillatory expansion. The non-physical stimulus may be heat, pH changes or redox changes. For example, the non-physical stimulus may be a change to physiological temperature or a change to physiological pH.

[0059] The hydrogel may undergo oscillatory stepwise expansion. Thus, it may be that the gel expands in steps. Stepwise expansion can be thought of as arising because the rate of expansion is oscillatory (i.e. relatively fast expansion, then a period in which the rate of expansion decreases, then a period of relatively little or no expansion, then a period in which the rate of expansion increases etc.). Thus, the gel may oscillate between periods of relatively rapid expansion and periods of no expansion or slow expansion. In these embodiments, the hydrogel will not revert to its original size between periods of expansion. This will typically occur in response to a change in conditions (the non-physical stimulus) that has an oscillatory rate of change. As illustrative examples, the hydrogel may expand in response to an increase in pH that has an oscillatory rate of change (oscillating between periods of relatively rapid increase in pH and periods of no increase or slow increase in pH) or the hydrogel may expand in response to a decrease in pH that has an oscillatory rate of change (oscillating between periods of relatively rapid decrease in pH and periods of no decrease or slow increase in pH).

[0060] The hydrogel may undergo oscillatory expansion and contraction. Thus, the hydrogel alternates between periods of expansion and periods of contraction. Where the hydrogel undergoes oscillatory expansion and contraction, the contraction will typically occur in response to a second non-physical stimulus that is the opposite of the nonphysical stimulus that causes the hydrogel to expand (the first non-physical stimulus). This will typically occur in response to a change of conditions that is itself oscillatory. As illustrative examples: the hydrogel may alternate between a period of expansion in response to an increase in pH and a period of contraction in response to a decrease in pH; the hydrogel may alternate between a period of expansion in response to a decrease in pH and a period of contraction in response to an increase in pH; the hydrogel may alternate between a period of expansion in response to the release of heat from the reaction and a period of contraction as the hydrogel cools; the hydrogel may alternate between a period of expansion in response to an increase in redox potential and a period of contraction in response to a decrease in redox potential; or the hydrogel may alternate between a period of expansion in response to a decrease in redox potential and a period of contraction in response to an increase in redox potential.

[0061] The catalyst may be suitable for catalysing an oscillatory oxidative carbonylation reaction of the organic compound. The catalyst may be selected such that, under the conditions of the oscillatory reaction, regeneration of the catalyst is autocatalytic. Autocatalytic means that reacted catalyst can only be regenerated in the presence of unreacted catalyst.

[0062] For the avoidance of doubt, in the context of the invention, an oxidative carbonylation may involve the reaction of an organic compound comprising an alkyne with a transition metal catalyst in the presence of a carbonylating agent (e.g. carbon monoxide), an organic alcohol and an oxidising agent (e.g. O2).

[0063] The oscillatory reaction may be a transition metal-catalysed oxidative carbonylation. The oscillatory reaction may be a palladium-catalysed oxidative carbonylation (PCOC) reaction. The oscillatory reaction may be a cobalt-catalysed oxidative carbonylation reaction. The alkyne of the organic compound may react with a carbonylating agent and an organic alcohol. For example, the alkyne of the organic compound may react with carbon monoxide and an organic alcohol. An illustrative PCOC reaction is below:

PhAc DMO Z-isomer E-isomer

[0064] The carbonylating agent may be selected from carbon monoxide, an aldehyde (e.g. phenylformate or pyridinyl methyl formate), a formamide (e.g. formanilide) or a carbonyl-containing metal complex (e.g. Rus(CO)i2 or Fe(CO)s). The carbonylating agent may be carbon monoxide.

[0065] The oscillatory reaction may be sustained for longer than 1 hour. The oscillatory reaction may be sustained for longer than 24 hours. The oscillatory reaction may be sustained for longer than 48 hours. The oscillatory reaction may be sustained for longer than 7 days. The oscillatory reaction may be sustained for longer than 28 days. The oscillatory reaction may be sustained for longer than 50 days.

[0066] It may be that the oscillatory or stepwise expansion of the hydrogel causes oscillatory release of the pharmaceutically active compound from the hydrogel.

[0067] Suitable oscillatory reactions are described in WO 2020/002909.

[0068] The composite material may be in a form suitable for oral administration. The composite material may be in the form of a powder. The composite material may be in the form of granules. The composite material may be in the form of pellets. Where the composite material is a powder, the powder may be compressed into a tablet or may be formulated in a capsule.

[0069] The composite material may be in a form suitable for agricultural administration. For example, the composite material may be formulated as a sustained release fertiliser.

[0070] The composite material of the invention may be a mucoadhesive agent in vivo. The composite material of the invention may be a mucoadhesive agent in vitro.

[0071] The composite material of the invention may be manufactured under mild conditions without the use of harsh chemicals. Thus, the manufacture of these composite materials of the invention may be facile, cheap and more environmentally friendly than prior art methods. [0072] The composite material of the invention is typically biodegradable. Thus, the composite materials of the invention may be environmentally sustainable.

Method of Making the Composite Material

[0073] The third aspect of the invention relates to a method of making a composite material, the method comprising: providing a mixture comprising a first polymer and a second polymer in a solvent, gelating the mixture to form a gelated mixture, and drying the gelated mixture to form the composite material.

[0074] The first polymer and second polymer may be as described in any of the embodiments in this specification relating to the first aspect or second aspect of the invention.

[0075] The solvent may be selected from: ethanol in water, chloroform in water, isopropanol in water, DMF in water, DMSO in water, acetonitrile in water, and acetone in water. The solvent may be ethanol in water.

[0076] The solvent may be ethanol in water in a concentration of from about 10% to about 60% v/v. The solvent may be ethanol in water in a concentration of from about 15% to about 50% v/v. The solvent may be ethanol in water in a concentration of from about 20% to about 40% v/v.

[0077] The mixture may comprise a solution of first polymer and a solution of second polymer in a ratio in the range of from about 1 :2 to about 2: 1 by volume, optionally in a range of about 1 :1.

[0078] The solution of first polymer may comprise the first polymer in an amount of from about 0.5% to about 5% by weight of the solution. The solution of first polymer may comprise the first polymer in an amount of from about 1 % to about 2% by weight of the solution, optionally from about 1.25% to 1.75% by weight of the solution. The solution of first polymer may comprise the first polymer in an amount of from about 1% to about 1.5% by weight of the solution, optionally about 1.35% by weight of the solution.

[0079] The solution of first polymer may further comprise a cross-linking agent in an amount of from about 0.001% to about 3.5% by weight of the solution, from about 0.01% to about 0.5% by weight of the solution, or from about 0.01 % to about 0.1 % by weight of the solution.

[0080] The solution of second polymer may comprise the second polymer in an amount of from about 30% to about 40% by weight of the solution.

[0081] The mixture may further comprise an effective amount of an active agent. The active agent may be a pharmaceutically active compound, or pharmaceutically acceptable salt thereof, and be present in a therapeutically effective amount. The pharmaceutically active compound, or pharmaceutically acceptable salt thereof, may be as described in any of the embodiments in this specification relating to the first aspect or second aspect of the invention. Alternatively, the active agent may be an agronomically active compound, or agronomically acceptable salt thereof, and be present in an agronomically effective amount. The agronomically active compound, or agronomically acceptable salt thereof, may be as described in any of the embodiments in this specification relating to the first aspect or second aspect of the invention.

[0082] The mixture may further comprise a pH regulator in an amount of about 0.1 — 2% by weight, optionally about 1% by weight. The pH regulator may be selected from: acetic acid, ascorbic acid, or other acids. Preferably, the pH regulator is acetic acid.

[0083] The mixture may further comprise an organic compound in an amount of about 5- 60% by weight and a transition metal catalyst in an amount of about 0.01-1% by weight. The organic compound and transition metal catalyst may be as described in any of the embodiments in this specification relating to the first or second aspect of the invention.

[0084] The step of providing the mixture comprising the first polymer and second polymer in a solvent may further comprise centrifuging the mixture. Alternatively or additionally, vacuum may be applied to the mixture comprising the first polymer and second polymer in a solvent. The vacuum may be applied once or multiple times successively.

[0085] The centrifuging may be performed in a mould. Alternatively or additionally, vacuum may be applied to fill the mould. The vacuum may be applied once or multiple times successively to fill the mould.

[0086] The step of gelating the mixture may be performed at a temperature in the range of from about 10°C to about 80°C. The step of gelating the mixture may be performed at a temperature in the range of from about 20°C to about 50°C. The step of gelating may be performed at a temperature of about 20°C, about 37°C or about 50°C.

[0087] The step of gelating the mixture may be performed at a temperature in the range of from about 10°C to about 80°C. The step of drying the gelated mixture may be performed at a temperature in the range of from about 20°C to about 50°C. The step of drying the gelated mixture may be performed at a temperature of about 20°C, about 37°C or about 50°C.

[0088] The step of gelating the mixture may be performed for at least about 24 hours.

The step of gelating the mixture may be performed for about 24 hours, about 48 hours, or about 72 hours. [0089] The step of drying the gelated mixture may be performed for at least about 24 hours. The step of drying the gelated mixture may be performed for about 24 hours, about 48 hours, or about 72 hours.

[0090] The steps of gelating the mixture and drying the gelated mixture may be performed at the same temperature (e.g. about 20°C, about 37°C or about 50°C). The step of gelating the mixture and drying the gelated mixture may be performed for the same length of time (e.g. about 24 hours, about 48 hours, or about 72 hours).

[0091] In a third aspect, the invention provides a composite material obtainable or obtained by the method of second aspect.

Transdermal Patch

[0092] The transdermal patch comprises a body and a plurality of microneedles attached to the body.

[0093] The plurality of microneedles comprise the composite material of the first, second aspect or fourth aspect. The body may comprise the composite material of the first aspect, second aspect or fourth aspect. The plurality of microneedles and the body may form a monolithic structure comprising the composite material of the first aspect, second aspect or fourth aspect.

[0094] Alternatively, the body may comprise a polymer selected from: cross-linked chitosan, cross-linked gelatin, cross-linked hyaluronan, cross-linked cellulosics, crosslinked polymethacrylates, cross-linked poloxamers, acrylic-vinylacetate, hydroxypropyl cellulose, dimeticone and mixtures thereof. The body may comprise a pouch.

[0095] The plurality of microneedles may be at least 50 pm in length, at least 100 pm in length, at least 200 pm in length, at least 300 pm in length, at least 400 pm in length, or at least 500 pm in length. It may be that the plurality of microneedles are from about 400 pm to about 900 pm in length. It may be that the plurality of microneedles are from about 400 pm to about 600 pm in length. It may be that the plurality of microneedles are about 500 pm in length.

[0096] The transdermal patch may further comprise a therapeutically effective amount of a pharmaceutically active compound, or pharmaceutically acceptable salt thereof.

[0097] The body of the transdermal patch may encapsulate a reservoir within which the pharmaceutically active compound, or pharmaceutically acceptable salt thereof, is retained. Additionally or alternatively, the pharmaceutically active compound, or pharmaceutically acceptable salt thereof, may be retained in the plurality of microneedles. Additionally or alternatively, the pharmaceutically active compound, or pharmaceutically acceptable salt thereof, may be uniformly distributed throughout the transdermal patch.

[0098] The reservoir may further comprise the organic compound comprising an alkyne, and/or the transition metal catalyst comprising palladium, platinum, iron, copper, cobalt, ruthenium, rhodium or iridium. The organic compound comprising an alkyne, and/or the transition metal catalyst comprising palladium, platinum, iron, copper, cobalt, ruthenium, rhodium or iridium may be uniformly distributed throughout the transdermal patch. Alternatively, the organic compound comprising an alkyne, and/or the transition metal catalyst comprising palladium, platinum, iron, copper, cobalt, ruthenium, rhodium or iridium may be uniformly distributed throughout the first polymer.

[0099] The transition metal catalyst may be suitable for catalysing an oscillatory oxidative carbonylation reaction of the organic compound.

[00100] The transition metal catalyst may comprise palladium. For example, the transition metal catalyst may comprise Pd ²⁺ , e.g. Pdh or PdCh. Alternatively, the transition metal catalyst may comprise cobalt. The transition metal catalyst may comprise cobalt ² *, e.g. CO(NO ₃ ) ₂ .6H ₂ O.

[00101] The transdermal patch may be configured to provide continuous release of the pharmaceutically active compound over a period of at least 1 minute, at least 1 hour, at least 24 hours, at least 48 hours, at least 7 days, at least 28 days, at least 40 days, or at least 50 days.

Implantable Device

[00102] The implantable device may further comprise a therapeutically effective amount of a pharmaceutically active compound, or pharmaceutically acceptable salt or solvate thereof.

[00103] The implantable device may be in the form of a disc. The disc may be a monolithic tablet comprising the composite material of the first aspect or third aspect.

[00104] The implantable device may be configured to provide continuous release of the pharmaceutically active compound over a period of at least 1 minute, at least 1 hour, at least 24 hours, at least 48 hours, at least 7 days, at least 28 days, at least 40 days, or at least 50 days.

[00105] The implantable device may be implantable subcutaneously, transdermally, rectally, intra-cranially, intra-uterinely, intra-articularly, intra-aurally, intra-ocularly and intra- gingivally.

Uses [00106] A sixth aspect of the invention provides a transdermal patch of the fourth aspect or an implantable device of the fifth aspect for use as a medicament. The transdermal patch or implantable device for use in said treatment typically comprises an effective amount of a pharmaceutically acceptable compound, or pharmaceutically acceptable salt thereof.

[00107] A seventh aspect of the invention provides a method of administering a pharmaceutical to a patient in need thereof, the method comprising applying a transdermal patch of the fourth aspect to the patient. The pharmaceutical may be suitable for the treatment or prevention of pain, arthritis, diabetes, asthma, cardiovascular disorders, cancer, Alzheimer’s disease, Parkinson’s disease and other neurological conditions.

[00108] An eighth aspect of the invention provides a method of administering a pharmaceutical to a patient in need thereof, the method comprising implanting the implantable device of the fifth aspect in the patient. The implantable device may be implanted subcutaneously, transdermally, rectally, intra-cranially, intra-uterinely, intraarticularly, intra-aurally, intra-ocularly and intra-gingivally, etc. The pharmaceutical may be suitable for the treatment or prevention of pain, arthritis, diabetes, asthma, cardiovascular disorders, cancer, Alzheimer’s disease, Parkinson’s disease and other neurological conditions.

[00109] A ninth aspect of the invention provides a use of the composite material of the first aspect or third aspect to administer a pharmaceutically active compound, or pharmaceutically acceptable salt thereof, to a patient. The pharmaceutically active compound may be suitable for the treatment of pain, arthritis, diabetes, asthma, cardiovascular disorders, cancer, Alzheimer’s disease, Parkinson’s disease and other neurological conditions, etc.

BRIEF DESCRIPTION OF THE DRAWINGS

[00110] Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which:

Figure 1 shows microneedles on a transdermal microneedle patch made of the composite material of the invention comprising a chitosan-genipin and PVP and including methylene blue (MB) as a model active agent.

Figure 2 shows a schematic representation of the structure of a composite material of the invention comprising a first polymer, a second polymer, and a cargo (e.g. an active agent or a fertiliser). The first polymer forms a mesh structure, which the second polymer and the cargo interpenetrate. Figure 3 shows an exemplary transdermal permeation profile from two different formulations (Formula 1 and Formula 2) of the transdermal microneedle patch of the invention. Formulas 1 and 2 contained different amounts of cargo (methylene blue). The permeation profile shows extra-long and tuneable drug release and permeation compared with the Conventional Formula (PVP transdermal microneedle patch). Skin permeation was assessed using a vertical Franz diffusion cell with an in vitro skin model (Merck Strat- M®) (right).

Figure 4 shows scanning electron microscopy (SEM) images of an exemplary transdermal microneedle patch of the invention. The images show the appearance of a microneedle before and after immersion in phosphate buffered saline (PBS; 0.01 M pH 7.4) for 48 hours, demonstrating the transition from a solid microneedle to a porous microneedle.

Figure 5 shows scanning electron microscopy (SEM) images showing the structural changes of microneedles employed in a transdermal patch of the invention comprising a chitosan-genipin and PVP. (A) Dry sample of the microneedles. (B) Microneedles after 1 hour in phosphate-buffered saline (PBS); (C) Microneedles after 6 hours in PBS; (D) Microneedles after 24 hours in PBS; (E) Microneedles after 48 hours in PBS.

Figure 6 shows puncture marks (stained dark blue) in a porcine skin model, confirming the ability of the microneedles comprising the composite material to overcome the stratum corneum barrier for transdermal drug delivery.

Figure 7 shows an exemplar SDS-PAGE gel showing Coomassie Brilliant Blue staining of supernatants from an exemplary release study of bovine serum albumin (BSA) from composite discs of the invention (Example 5). The sole protein detected in any sample was between 63 kDa and 75 kDa, which confirmed the identity of the protein as BSA (66 kDa).

Figure 8 shows bovine serum albumin (BSA) release from a composite disc formulation of the invention, compared with a PVP disc formulation, a wet hydrogel formulation and a dried hydrogel formulation. All formulations contained 10 mg of BSA. Error bars represent standard deviation (n = 3) and are shown assymetrically to avoid overlaps, for clarity.

Figure 9 should the fluorescence intensity change over time with varying BSA concentrations (0.2 to 50 mg/mL) in the presence of a constant genipin concentration (0.5 mg/mL) in microneedles of the disclosure. Figure 10 shows the mechanical strength of microneedles loaded with dexSP in terms of the axial failure force under compression. Data was obtained using a texture analyser (mean ± SD, n = 15). A comparative benchmark for the typical failure force for microneedles of polylactic acid is provided, said microneedles having a similar size (1 mm long, 0.23 mm wide at base) to the microneedles of the example.

Figure 11 shows the drug release profile of dexSP (mean ± SD; n = 5) from microneedles of the invention. Some transformation of dexSP into its parent drug dexamethasone (dex) was detected.

Figure 12 shows the effect of dexSP released from microneedles of the disclosure on the production of TNF-alpha human peripheral blood mononuclear cells (PBMC) at various time points, in the presence and absence of the endotoxin lipopolysaccharide (LPS). Drug release media from DexSP-loaded microneedles (ll-Dex) and blank microneedles without DexSP (Il-Blank) was added to PBMC +/- 100ng/mL LPS for 19 hours. TNF-alpha production was measured using an enzyme-linked immunosorbent assay (ELISA). This experiment was carried out on PBMC from one donor in triplicate (n = 3). Statistical analysis was performed using one-way ANOVA with post-hoc Tukey testing. **** p < 0.0001.

DETAILED DESCRIPTION

[00111] Throughout this specification, the term ‘oscillatory’ is intended to mean repetitive variation in a property over time, i.e. alternation between periods of high or low values for said property. Examples of said property include (rate of release of an active agent, size (i.e. degree of expansion) of gel, pH, temperature, rate of expansion, rate of pH change, rate of change of temperature, rate of reaction, rate of change of rate of reaction). It is not intended to mean a mathematically precise sinusoidal pattern, merely a repeated alternation between a relative peak and a relative trough for said property. A gel comprises a solid phase and a liquid phase. The liquid phase is distributed throughout the solid phase.

[00112] The term “polymer” used herein may refer to a single species of polymer or to a polymeric mixture comprising multiple species of polymer blended together to create a new material with different physical properties to each individual species of polymer.

[00113] The term “hydrogel” refers to a hydrophilic cross-linked polymer comprising water. A hydrogel may comprise at least about 20% w/w water. For example, a hydrogel may comprise at least about 30% w/w water, at least 50% w/w water, at least 70% w/w water, at least 90% w/w water, at least 95% w/w water or at least 99% w/w water. [00114] The term “biocompatible” refers to a component that does not stimulate a response or stimulates only a mild and/or transient response, as opposed to a severe or escalating response, when placed or implanted into the human or animal body, or when administered to a plant.

[00115] The term “active agent” may refer to any pharmaceutically or agronomically active compound. The active agent may be a small molecule. The active agent may be a biopolymeric drug, e.g. protein, an antibody or conjugate thereof, a polysaccharide, a polynucleotide. The active agent may be a fertiliser, a fungicide, a pesticide or an insecticide.

[00116] The term ‘non-physical stimulus’ is intended to describe any change in the chemical or energetic conditions to which the hydrogel is subjected. This change in conditions will be generated by the oscillatory reaction occurring within the hydrogel. The term ‘non-physical stimulus’ is intended to exclude the use of a solid object or externally applied pressure change to cause the hydrogel to expand and contract. The non-physical stimulus may be a change in pH (e.g. an increase in pH or a decrease in pH). The nonphysical stimulus may be a change in temperature (e.g. an increase in temperature). The non-physical stimulus may be a change in redox potential (e.g. an increase in redox potential or a decrease in redox potential).

[00117] The oscillatory reaction is sustained without any further catalyst being added. Sustained means that the oscillatory nature of the reaction (and the resultant effect, e.g. stepwise expansion or expansion and contraction of the hydrogel or the release of an active agent) is continuous over long periods of time (e.g. greater than 1 hour) without any need to add catalyst. Typically, the reaction will continue until substantially all of the organic compound has reacted. In this context, the term ‘substantially all’ may mean that greater than 50%, e.g. greater than 75% or greater than 90% of the organic compound has reacted. The oscillatory reaction will typically also be sustained without the need to add further organic compound. The reaction may be described as a batch-like reaction, at least with respect to the catalyst and the organic compound. It may be that a supply of other reactants, e.g. CO or alcohol, is available to the hydrogel in order to sustain the oscillatory reaction.

[00118] An organic compound is a group of atoms that comprise carbon and hydrogen and that are covalently bonded together. The organic compound may also comprise one or more heteroatoms selected from oxygen, nitrogen, sulfur, phosphorous, halogen and boron. The organic compound may be covalently attached to a component of the polymeric solid phase. In this instance, the organic compound is a part of the larger macromolecule that comprises both the polymer chain and one or more of the groups of covalently bonded atoms that are herein referred to as the organic compound.

[00119] The term “pharmaceutically acceptable salts” is meant to include salts of the pharmaceutically active compounds which are prepared with relatively nontoxic acids or bases, depending on the particular substituents found on the compounds described herein. When compounds contain relatively acidic functionalities, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, or magnesium salt, or a similar salt. When compounds contain relatively basic functionalities, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids and the like, as well as the salts derived from relatively nontoxic organic acids like acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Also included are salts of amino acids such as arginate and the like, and salts of organic acids like glucuronic or galactunoric acids and the like (see, for example, Berge et al., “Pharmaceutical Salts”, Journal of Pharmaceutical Science, 1977, 66, 1-19). Certain specific compounds of the present invention contain both basic and acidic functionalities that allow the compounds to be converted into either base or acid addition salts.

[00120] Thus, the compounds of the present disclosure may exist as salts with pharmaceutically acceptable acids. The present invention includes such salts. Examples of such salts include hydrochlorides, hydrobromides, sulfates, methanesulfonates, nitrates, maleates, acetates, citrates, fumarates, tartrates (e.g. (+)-tartrates, (-)-tartrates or mixtures thereof including racemic mixtures, succinates, benzoates and salts with amino acids such as glutamic acid. These salts may be prepared by methods known to those skilled in the art.

[00121] The term “agronomically acceptable salts” is meant to include salts of the agronomically active compounds which are prepared with relatively non-phytotoxic acids or bases, depending on the particular substituents found on the compounds described herein. Suitable salts include, but are not limited to, salts of acceptable inorganic acids such as hydrochloric, sulfuric, phosphoric, nitric, carbonic, boric, sulfamic, and hydrobromic acids, or salts of agronomically acceptable organic acids such as acetic, propionic, butyric, tartaric, maleic, hydroxymaleic, fumaric, malic, citric, lactic, mucic, gluconic, benzoic, succinic, oxalic, phenylacetic, methanesulfonic, toluenesulfonic, benzenesulfonic, salicylic, sulfanilic, aspartic, glutamic, edetic, stearic, palmitic, oleic, lauric, pantothenic, tannic, ascorbic and valeric acids. Suitable salts also include salts of inorganic and organic bases, e.g. counterions such as Na, Ca, K, Li, Mg, ammonium, trimethylsulfonium. The compounds may also be obtained, stored and/or used in the form of an N-oxide.

[00122] The phrase “effective amount” refers to an amount sufficient to attain the desired result. The phrase “therapeutically effective amount” means an amount sufficient to produce the desired therapeutic result. Generally, the therapeutic result is an objective or subjective improvement of a disease or condition, achieved by inducing or enhancing a physiological process, blocking or inhibiting a physiological process, or in general terms performing a biological function that helps in or contributes to the elimination or abatement of the disease or condition.

[00123] The invention concerns amongst other things the treatment of a disease. The term “treatment”, and the therapies encompassed by this invention, include the following and combinations thereof: (1) hindering, e.g. delaying initiation and/or progression of, an event, state, disorder or condition, for example arresting, reducing or delaying the development of the event, state, disorder or condition, or a relapse thereof in case of maintenance treatment or secondary prophylaxis, or of at least one clinical or subclinical symptom thereof; (2) preventing or delaying the appearance of clinical symptoms of an event, state, disorder or condition developing in an animal (e.g. human) that may be afflicted with or predisposed to the state, disorder or condition but does not yet experience or display clinical or subclinical symptoms of the state, disorder or condition; and/or (3) relieving and/or curing an event, state, disorder or condition (e.g., causing regression of the event, state, disorder or condition or at least one of its clinical or subclinical symptoms, curing a patient or putting a patient into remission). The benefit to a patient to be treated may be either statistically significant or at least perceptible to the patient or to the physician. It will be understood that a medicament will not necessarily produce a clinical effect in each patient to whom it is administered; thus, in any individual patient or even in a particular patient population, a treatment may fail or be successful only in part, and the meanings of the terms “treatment” and “prophylaxis” and of cognate terms are to be understood accordingly. The compositions and methods described herein are of use for therapy and/or prophylaxis of the mentioned conditions.

[00124] Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of them mean “including but not limited to”, and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps. Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

[00125] Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive. The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

[00126] The reader's attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.

EXAMPLES

Example 1 : General procedure for making composite material

[00127] The raw ingredients of crosslinked genipin-chitosan and PVP (and optionally a therapeutic agent and/or any other excipient, e.g. a pH regulator such as acetic acid) were dissolved in a common/mutual solvent (e.g. 20% v/v, 30% v/v or 40% v/v ethanol in water). The mixture was placed in a container and allowed to gelate at an appropriate temperature (e.g. 20°C, 37°C or 50°C), then dried at an appropriate temperature (e.g. 20°C, 37°C or 50°C) to form the composite material. The gelation time and drying time can vary depending on the properties of the ingredients used (e.g. the therapeutic agent, polymer 1 and polymer 2), taking into account the stability of ingredients under the manufacturing conditions. Typically, this will be 24 hours, 48 hours or 72 hours for each of the gelation step and drying step.

[00128] A mixture of either 30% or 40% PVP solution (in 20:80% v/v ethanokwater as the solvent) was mixed in a ratio of 1:1 with the hydrogel (HG) solution (1.35% chitosan and

0.1% genipin dissolved in 1% acetic acid solution). Approximately 0.8 ml of the formed aqueous PVP polymer blend was loaded in each MN array mould. The moulds were then centrifuged at 4000 rpm for 50 min to ensure complete filling of the moulds. Alternatively or additionally, vacuum can be applied once or multiple times successively to fill the moulds. The moulds were then wrapped in Parafilm® M to prevent premature drying and incubated at 37°C for 24 h to allow gelation and then the parafilm was removed and the composite MN arrays were allowed to dry at RT for 48 hours before demoulding.

Physical Characterisation

[00129] The microneedles remain intact over 6 days in phosphate-buffered saline (PBS). Without wishing to be bound by theory, it is thought that whatever PVP has dissolved in the buffer solution is replaced by the buffer itself, retaining the pores in the chitosan- genipin hydrogel matrix.

[00130] Figure 5 shows the progressive changes in the composite material of the microneedles in an aqueous environment (PBS) where the soluble PVP component dissolves away, leaving behind the insoluble chitosan-genipin component. discs

[00131] The polymer blends were prepared as Example 2 above. Once formed, 1 ml of the polymer blend was poured into cylindrical vials (diameter up to 1 cm) and centrifuged at 4000 rpm for 5 min. Alternatively or additionally, vacuum can be applied once or multiple times successively. The vials were wrapped in Parafilm and incubated at 37°C for 24 h to allow the gelation and then the parafilm was removed and the vials were opened and left to dry at RT for 48 hours. the MN arrays and composite discs with blue as a model active agent

[00132] MB was dissolved in deionized water at 20 mg/ml and an appropriate volume of the MB solution was mixed with the content of each MN array or disc before the centrifugation step to load 0.5-10 mg MB in each MN array or disc (for example, 100 pl of the 20 mg/ml MB solution was loaded in each MN array or disc, to get 2 mg MB per MN array or disc). Example 5: Release of a model protein cargo from composite discs

[00133] Bovine serum albumin (BSA; ~66 kDa) was used as a model protein drug to demonstrate protein loading and release from composite discs of the invention. The discs were prepared by first mixing stock solutions of chitosan (1.9% w/v in 1% aqueous acetic acid solution), PVP (40% w/v in 80%:20% deionised water: ethanol), genipin (1% w/v in deionised water) and BSA (10% w/v in deionised water).

[00134] The different formulas were prepared by mixing the stock solutions according to Table 1.

Table 1: Formulation of BSA in the composite and other polymer discs

[00135] Vials containing 1 mL of the formulations were wrapped in Parafilm® M to prevent solvent evaporation and incubated at 37°C for 24 hours to allow gelation. The Parafilm® M was then removed for all the formulations, except the wet hydrogel, and the vials were left open to dry at room temperature (~20°C) for 48-72 hours until use. Meanwhile, the wet hydrogels were kept in the fridge (4°C), well-sealed in Parafilm® M, until use.

[00136] Formulations loaded with 10 mg BSA were placed in 4 mL PBS (pH 7.4) and maintained at 32 °C in a water bath throughout the experiment. The PBS was aspirated at predefined time intervals for analysis) and replaced with an equal volume of fresh PBS to maintain sink conditions. The supernatant was sampled at defined intervals and analysed by the Bradford assay to determine the amount of protein content released from the discs. The identity of the protein was confirmed with Coomassie brilliant blue staining and Western blotting.

Coomassie Brilliant Blue staining

[00137] At the end of the release study, equal amounts of protein (1.5 pg) from each endpoint sample, as well as BSA standard solutions (prepared in PBS, BSA concentrations ranging from 0.025-0.8 mg/mL), were analysed by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), which separated proteins according to their molecular weights. Following electrophoresis, the proteins were stained using Coomassie Brilliant Blue for 30 minutes before de-staining overnight in a mixture of water: methanol :acetic acid (volume ratio: 5:4:1). This was followed by scanning using Syngene Ingenius image scanner.

Western Blotting

[00138] Western blotting was performed essentially as previously described (Abdelghany et al., 2020, https ://do i org/10.1016/j fct.2020 111593). Briefly, the amount of the albumin released was assessed using the Western blot assay. After protein solutions were extracted from the samples at different time intervals, equal amounts of proteins were analysed by SDS-PAGE, which separated proteins according to their molecular weights. Following electrophoresis, the proteins were transferred to a nitrocellulose membrane (Amersham Bioscience, Piscataway, NJ, USA) using wet transfer. Non-specific binding sites were blocked by placing the membranes in 5% skim milk. The membranes were then incubated with the albumin antibody solution (1 :500; Thermo Fisher Scientific, MA, USA) overnight on a roller shaker at 4°C. Subsequently, they were washed and incubated with the horseradish peroxidase (HRP)-conjugated secondary antibody (1 :1000; Fluka, St. Louis, MO, USA). Finally, the blots were developed with chemiluminescence detection reagents. Chemiluminescence detection of the HRP activity (equivalent for the target protein position and amount) on the membrane was performed with the Pierce ECL Western Blotting Substrate (Thermo Scientific, UK). The membrane was fixed into a cassette and exposed to x-ray film in a dark room followed by the development of x-ray film with an X-omat developer (Kodak).

Results

[00139] For each formulation, the total amount of protein released increased as time progressed. SDS-PAGE with Coomassie Brilliant Blue staining and Western blot confirmed that the only protein detected in the samples (supernatants) was between 63-75 kDa (Figure 7). This confirmed that the protein was BSA (66 kDa). BSA release from the PVP disc was complete within 1 hour. The composite formulation significantly prolonged BSA release for at least 18 days (Figure 8). At 18 days, 69.8% (s.d. = 12.5%) of the BSA dose had been released from the composite formulation. This release rate was higher than those from the wet and dried hydrogel formulations, but significantly slower than that from the PVP formulation.

Example 6: Interaction of model protein cargo with components of MN array [00140] Bovine serum albumin (BSA; ~66 kDa) was used as a model protein drug to study its interaction with the cross-linking agent in the microneedle arrays of the invention. The BSA-loaded microneedle arrays were prepared as described in Examples 2 and 4. The concentration of BSA used in the production of the microneedle arrays was varied from 0.2 mg/mL to 50 mg/mL. The interaction between BSA and genipin in each of microneedle arrays was assessed over 24 hours by analysing the fluorescence intensity (Figure 9).

[00141] For each of the concentrations of BSA, the interaction between genipin and BSA was delayed by at least one hour. Without wishing to be bound by theory, it is thought that the crosslinking between chitosan and genipin (i.e. network formation) took priority over that between BSA and genipin, leaving the BSA interpenetrated in the hydrogel network ready for release upon administration. At concentrations <5mg/mL, the reaction between protein and genipin was delayed by 2 hours or more.

Example 7: Loading MN arrays with dexamethasone sodium phosphate (dexSP) as an exemplary active agent

[00142] dexSP was loaded into the MN array following the same procedure as described for MB in Example 4.

Mechanical Strength

[00143] The strength of the dexSP loaded microneedles was determined via texture analysis of microneedles having different loadings, whereby the axial failure force of the microneedle under compression was measured (Figure 10). Each of the microneedles loaded with 3 mg dexSP, 6 mg dexSP and no dexSP achieved a mean axial failure force at least 0.6 N higher than that reguired for skin penetration.

Release of dexSP

[00144] Drug release of dexSP from the microneedle array (3 mg loading) in PBS was demonstrated over 80 days (Figure 11). After day 28, some conversion into dexamethasone (dex, the parent compound of dexSP) was detected. Both dexSP and dex are pharmacologically active. The dexSP release study was performed across a synthetic membrane (Parafilm M with polyethylene cling film underlay) in Franz diffusion cells, at 32°C. The receptor fluid was 10mM phosphate buffered saline (pH 7.4). The active agents dexSP and dex were guantified by high-performance liguid chromatography (HPLC).

Biocompatibility

[00145] Biocompatibility and pharmacological action of microneedle-encapsulated dexamethasone sodium phosphate (DexSP) was examined in peripheral blood mononuclear cells (PBMC), which consists of immune cells sensitive to immunomodulatory agents, such as bacterial endotoxin (e.g., lipopolysaccharide = LPS, used in this study as a positive control/proinflammatory agent, 100 ng/mL) and other proinflam matory/cytotoxic chemicals (Figure 12).

[00146] The receptor fluid from Franz diffusion cells (FDC), containing substances released from the microneedles on days 3, 14, 35 and 59, were incubated with PBMC from 1 donor in triplicate (n = 3). Cellular response was characterised in terms of TNF-a release (a pro-inflammatory cytokine) from the PBMC. Lower TNF-a levels indicate less immune activation/inflammation and, in terms of chemical substances, a better biocompatibility profile.

[00147] The controls used in the biocompatibility testing were:

Untreated = Normal cell culture conditions, with no treatment/intervention

+LPS = Endotoxin added to culture media

DexSP + LPS = Endotoxin and DexSP added to culture media.

[00148] As indicated in Figure 12, the PBMC culture was activated by LPS, while dexSP suppressed immune activation of PBMC by LPS. FDC receptor fluid from blank microneedles (U-Blank) showed low levels of immune activation, comparable to LPS immune activation suppressed by dexSP/dex (DexSP + LPS).

[00149] U-Blank + LPS led to immune activation of the PBMC, in all cases almost doubling the TNF-a release from the PBMC compared to U-Blank without LPS. This verifies that the PBMC were responsive to LPS.

[00150] LPS activated the PBMC to a lesser extent in FDC receptors fluids taken from blank microneedles (U-Blank + LPS) compared to the +LPS control. This suggests a possible anti-inflammatory effect of materials released from the microneedles, and/or that low-level activation by the blank sample could have affected the ability of the cells to further respond to LPS to the same extent.

[00151] FDC receptor fluid from microneedles containing dexSP (U-Dex) showed an effective anti-inflammatory effect. In particular, microneedles containing DexSP (U-Dex) effectively suppressed the low-level inflammation and returned TNF-a to a comparable level as the untreated sample.

[00152] Microneedles containing DexSP effectively suppressed TNF-a release in response to LPS (U-Dex + LPS). This demonstrates that the DexSP released from the microneedles was pharmacologically functional/active.