Skin permeabilisation device

Abstract

The invention provides a skin permeabilisation device, a method of obtaining interstitial fluid from a skin tissue surface of a subject, and a method of delivering an agent to the skin tissue surface of a subject. An exemplary skin permeabilisation device includes a chamber that can form a hypobaric seal with a skin tissue surface to provide a permeabilisation surface sealed within the chamber, and at least one protrusion located within the chamber that contacts a majority of the permeabilisation surface, thereby restrict doming of the permeabilisation surface.

Description

[0001] Skin permeabilisation device

[0002] Field of Invention

[0003] The present invention relates to a skin permeabilisation device, a method of obtaining interstitial fluid from a skin tissue surface of a subject, and a method of delivering an agent to the skin tissue surface of a subject.

[0004] Background to the Invention

[0005] The human skin forms the perfect unsound biological barrier. It allows chemical ingress to the extent required to sense changes in the external environment and sufficient chemical egress such as to maintain the tissue's, physiological, biomechanical, and barrier functions. The semi-permeable nature of the skin cannot be assigned to a single identifiable pathway of molecular exchange between the external and internal compartments of the human body. Rather, chemical transport across the skin must be understood in its physiological context, which is a choreographed interplay of different pathways that work together to mediate movement both into and out of the tissue. Therefore, the natural movement of chemicals across the skin barrier is typically insufficient to allow drugs to be delivered into the tissue easily or molecules to be directly extracted out of it.

[0006] There remains a significant challenge with delivering molecules into the skin and extracting important chemicals from the skin that can inform us about health and disease in the body. When these chemical signals are used in disease diagnosis, therapy selection, treatment monitoring and clinical outcome profiling, they are often termed biomarkers. Biomarkers can be extracted from the blood, but this results in a 1000-1500-fold dilution of chemical signals secreted by cells (1). Interstitial fluid (ISF), a non-clotting fluid that surrounds cells, is also a rich source of biomarkers. It contains 98% of the biomarkers found in the blood, but because cell secretions undergo less dilution in this fluid, it also contains numerous unique chemical signals (up to 12% of its total signals compared to the blood) (2).

[0007] Typical methods to collect ISF - suction blistering (3), punch biopsy (4), microneedles (2), and tape stripping - are invasive and all cause some degree of skin damage, with the least invasive methods collecting the lowest levels of biomarkers. For example, in the recently completed SOFTER trial, tape strips were used to profile skin cytokines in babies, but they yielded only very low signals (unpublished). Therefore, it remains challenging to use these techniques to extract ISF biomarkers in numerous clinical scenarios.

[0008] The inventors previously developed a method involving local application of controlled topical hypobaric pressure to achieve needle-free permeabilization to allow delivery of drugs into the skin. Preliminary work used 500 mBar of hypobaric pressure to stretch the skin, which thins the tissue by 20%, opens the hair follicles by 30% or more, and increases the skin blood flow underneath the chamber by 8.5% (5).

[0009] However, there remains a need to provide a method of permeabilising the skin in order to extract ISF and / or deliver agents in a non-invasive manner which avoids or minimises damage to the tissue.

[0010] Summary of the Invention

[0011] According to a first aspect, there is provided a skin permeabilisation device comprising: a chamber to be placed in contact with a skin tissue surface of a subject and capable of forming a hypobaric seal with the skin tissue surface to provide a permeabilisation surface sealed within the chamber; at least one protrusion positioned within the chamber and arranged to contact a majority of the permeabilisation surface to restrict doming of the permeabilisation surface.

[0012] The skin permeabilisation device according to the invention is an optimised device which restricts doming (i.e., parabolic deformation) of the skin tissue surface in response to hypobaric pressure. In other words, the skin tissue is prevented or limited from stretching vertically or upwardly (out of plane of the skin tissue surface), and promoted to stretch laterally (within the plane of the skin tissue surface), owing to the contact of the protrusion against the skin tissue surface. As a result, damage to the tissue is minimised or abolished. This is a crucial advantage over the prior art devices which all exert some form of damage to the skin, resulting in pain for the subject and a disrupted tissue barrier.

[0013] In addition, the inventors have demonstrated that significantly more ISF can be extracted using the device according to the invention (e.g., 2-3 fold more; in one example 2-4 pl in total extracted in one sample) in contrast to prior art devices. This provides the advantage that fewer samples need to be obtained from the subject to provide a certain volume of ISF for subsequent analysis of biomarkers. In addition, the inventors have shown that larger molecules within the ISF can be extracted using their device, increasing the accuracy and applicability of the sampling. This widens the clinical use of ISF samples, and a greater number of biomarkers can be extracted using the device. Furthermore, the device has flexibility in sample collection as the ISF can be extracted in the presence or absence of an extraction fluid. A further advantage is that the sample can be collected without being contaminated by blood, as is the case when needles and microneedles are used.

[0014] As used herein, the term "interstitial fluid" or "ISF" means the substantially clear, substantially colourless fluid which occupies the space between cells.

[0015] The term "skin permeabilisation device", as used herein, refers to a device which can be used to permeabilise the skin tissue barrier to facilitate delivery of molecules to the skin tissue and / or extraction of ISF from the skin tissue. In some embodiments, the skin permeabilisation device is for extraction of skin ISF from the skin tissue (i.e., a skin ISF extraction device). The skilled person will recognise that the fluid extracted from the skin tissue surface may be a mixture of skin ISF and sweat. In some embodiments, the skin permeabilisation device is for delivery of an agent to the skin tissue (i.e., an agent delivery device, such as a drug delivery device or a biomoiecuie delivery device).

[0016] The chamber may be any suitable chamber that can be placed in contact with a skin tissue surface, can form a hypobaric seal with the skin tissue surface and can receive ISF extracted from the permeabilisation surface and / or receive a fluid to be subsequently delivered to the skin tissue. In other words, the chamber can be applied to the skin tissue surface and a negative pressure can be applied to the chamber, sealing the chamber to the skin tissue surface and resulting in the extraction of ISF from the skin tissue and / or the delivery of a fluid to the skin tissue. The skin tissue surface located within the chamber is referred to as the "permeabilisation surface", as this is the surface which is permeabilised as a result of the application of negative pressure to allow ISF extraction from the skin tissue and / or fluid delivery to the skin tissue.

[0017] The chamber may be referred to as a "negative pressure chamber" or a "vacuum chamber", and the skilled person will recognise that any suitable shape, material and dimensions of the chamber may be employed provided that the chamber can carry out its function of forming a hypobaric seal with the skin tissue surface when negative pressure is applied and receiving fluid (i.e. extracted ISF and / or fluid to be delivered).

[0018] In some embodiments, the chamber is substantially cylindrical, cuboidal, hemispherical or a frustum. In some embodiments, the chamber is substantially cylindrical. The skilled person will appreciate that the dimensions of the chamber may be selected to suit a particular subject group. As an example, the dimensions of the chamber may be 0.5-10 cm for an adult human subject. As a further example, the dimensions of the chamber may be 0.5-3 cm for an infant human subject. As a further example, the dimensions of the chamber may be 0.5-2 cm for a mouse or 0.5-5 cm for a rat.

[0019] In some embodiments, the device further comprises a means for generating negative pressure within the chamber to form the hypobaric seal. For example, the device may further comprise a vacuum pump (and optionally, associated fluidic channels). However, the skilled person will recognise that various methods are known for generating negative pressure within a chamber and any suitable method may be employed.

[0020] In some embodiments, the chamber comprises a sealable lid or port through which the interior of the chamber can be accessed after the device is applied to the skin tissue surface. As an example, the chamber may comprise a sealable lid which can be closed to allow the hypobaric seal to be formed, and which can be opened to access the interior of the chamber (e.g., to withdraw extracted ISF and / or to add an agent to be delivered). Fluid (e.g., ISF, extraction fluid, or delivery agent) may be added or withdrawn from the chamber in any suitable manner, for example, using a pipette.

[0021] In some embodiments, the chamber is arranged to receive extraction fluid (e.g., saline) or an absorbent substrate (e.g., gauze, pad or mesh). In some embodiments, the chamber is arranged to receive a mesh, such as a honeycomb mesh. The term "extraction fluid", is intended to mean a fluid which can be added to the chamber into which the ISF is extracted. The extraction fluid (including the extracted ISF) can be subsequently collected from the chamber.

[0022] The skin tissue surface may be any suitable skin tissue surface intended to be permeabilised (for example to extract ISF and / or deliver an agent). In some embodiments, the skin tissue surface is arm skin. In some embodiments, the skin tissue surface is forearm skin.

[0023] In some embodiments, the skin tissue surface is unbroken tissue (i.e., the skin tissue has not been penetrated by a foreign object, such as a needle, microneedle or bore).

[0024] The subject may be any suitable subject from which it is desired to permeabilise skin (for example to extract ISF and / or deliver an agent). In some embodiments, the subject is an animal. In particular embodiments, the subject is a mammal, such as a human, non-human primate, dog, pig, cow, rat, mouse, guinea pig or rabbit. In some embodiments, the subject is a human or a rodent. In some embodiments, the subject is a human.

[0025] At least one protrusion is positioned within the chamber and arranged to contact the permeabilisation surface to restrict doming of the permeabilisation surface. In other words, the contact of the at least one protrusion against the permeabilisation surface prevents or limits the permeabilisation surface from stretching vertically or upwardly (out of plane of the skin tissue surface). It will be appreciated by the skilled person that the at least one protrusion does not necessarily contact the permeabilisation surface when the device is initially applied to the skin (before negative pressure is applied), but does contact the permeabilisation surface when negative pressure is applied and the hypobaric seal is formed. In other embodiments, the at least one protrusion contacts the permeabilisation surface before negative pressure is applied. The term "arranged to contact the permeabilisation surface", as used herein, refers to direct contact of the protrusion against the skin, as well as indirect contact of the protrusion against the skin, such as through an absorbent gauze, pad or mesh.

[0026] The skilled person will appreciate that any suitable shape, material and dimensions of the at least one protrusion may be employed provided that the at least one protrusion can carry out its function of contacting the permeabilisation surface to restrict doming of the permeabilisation surface.

[0027] In some embodiments, the at least one protrusion is substantially cylindrical, cuboidal, hemispherical or a frustum. In some embodiments, the at least one protrusion is substantially cylindrical. In some embodiments, the at least one protrusion is a pillar or a post. In some embodiments, the at least one protrusion is a solid shape. In some embodiments, the at least one protrusion occupies a majority of the volume of the chamber.

[0028] In some embodiments, the at least one protrusion is made of the same material as the chamber. In some embodiments, the at least one protrusion is made of a different material from the chamber. In some embodiments, the at least one protrusion and the chamber are integrally formed. In some embodiments, the at least one protrusion functions as a sealable lid of the chamber (i.e., the protrusion is a lid which can be removed to access the interior of the chamber, and can be replaced to seal the chamber). In some embodiments, the at least one protrusion and the chamber are coaxial. In some embodiments, the at least one protrusion is located in the centre of the chamber.

[0029] In some embodiments, the at least one protrusion is non-movable. The term "non-movable", as used herein, means that the at least one protrusion does not move with respect to the chamber. In certain embodiments, the at least one protrusion is not capable of reciprocating motion within the chamber (i.e., acting as a piston). In some embodiments, the at least one protrusion is not retractable (i.e., the protrusion cannot be partially or fully withdrawn from the chamber).

[0030] In some embodiments, the at least one protrusion comprises a contact face that is arranged to contact the permeabilisation surface to restrict doming of the permeabilisation surface. In some embodiments, the contact face is circular. In some embodiments, the contact face is a continuous surface (i.e., without holes). In some embodiments, the contact face is arranged to contact at least the centre of the permeabilisation surface.

[0031] In some embodiments, the contact face has a longest dimension of from about 5 mm to about 100 mm. In some embodiments, the contact face has a longest dimension of from about 10 mm to about 90 mm. In some embodiments, the contact face has a longest dimension of from about 20 mm to about 80 mm. In some embodiments, the contact face has a longest dimension of from about 30 mm to about 70 mm. In some embodiments, the contact face has a longest dimension of from about 40 mm to about 60 mm. In some embodiments, the contact face has a diameter of from about 5 mm to about 100 mm. In some embodiments, the contact face has a diameter of from about 10 mm to about 90 mm. In some embodiments, the contact face has a diameter of from about 20 mm to about 80 mm. In some embodiments, the contact face has a diameter of from about 30 mm to about 70 mm. In some embodiments, the contact face has a diameter of from about 40 mm to about 60 mm.

[0032] In some embodiments, the contact face has a longest dimension of from about 10 mm to about 20 mm. In some embodiments, the contact face has a longest dimension of from about 12 mm to about 18 mm. In some embodiments, the contact face has a longest dimension of from about 14 mm to about 16 mm. In some embodiments, the contact face has a diameter of from about 10 mm to about 20 mm. In some embodiments, the contact face has a diameter of from about 12 mm to about 18 mm. In some embodiments, the contact face has a diameter of from about 14 mm to about 16 mm.

[0033] In some embodiments, the longest dimension or diameter of the contact face is from about 60 to about 95% of the longest dimension or diameter of the chamber. In some embodiments, the longest dimension or diameter of the contact face is from about 70 to about 90% of the longest dimension or diameter of the chamber. In some embodiments, the longest dimension or diameter of the contact face is from about 80 to about 85% of the longest dimension or diameter of the chamber.

[0034] In some embodiments, the contact face comprises a relief pattern. In some embodiments, the contact face comprises a relief pattern at or near to the outer edges of the contact face. In some embodiments, the contact face comprises a relief pattern of concentric shapes. In some embodiments, the contact face comprises a relief pattern of concentric circles. In some embodiments, the contact face comprises a relief pattern of concentric circles at or near to the outer edges of the contact face. The relief pattern provides the advantage of reducing pinching of the skin tissue surface.

[0035] In some embodiments, the at least one protrusion does not extend beyond the chamber (i.e., the at least one protrusion is entirely contained within the chamber). As an example, the contact face may be positioned within the chamber. In some embodiments, the at least one protrusion comprises at least two protrusions. In some embodiments, the at least one protrusion comprises at least three protrusions, In some embodiments, the at least one protrusion comprises at least four protrusions. In some embodiments, the at least one protrusion comprises at least five protrusions. In particular embodiments, the device comprises a single protrusion.

[0036] In some embodiments, the at least one protrusion comprises a reservoir (e.g., arranged to receive an agent for delivery to the skin).

[0037] The restriction in doming of the permeabilisation surface may be quantified by any using any suitable means, for example in terms of an absolute or relative reduction of the maximum height of the domed surface (i.e., defined from the plane of the skin tissue surface) or an absolute or relative reduction of the volume of the domed area (i.e., defined from the plane of the skin tissue surface) or an absolute or relative reduction of the surface area of the permeabilisation surface subjected to doming.

[0038] In some embodiments, doming of the permeabilisation surface is restricted by at least 50% in terms of a reduction of the maximum height of the domed surface. In some embodiments, doming of the permeabilisation surface is restricted by at least 60% in terms of a reduction of the maximum height of the domed surface. In some embodiments, doming of the permeabilisation surface is restricted by at least 70% in terms of a reduction of the maximum height of the domed surface. In some embodiments, doming of the permeabilisation surface is restricted by at least 80% in terms of a reduction of the maximum height of the domed surface. In some embodiments, doming of the permeabilisation surface is restricted by at least 90% in terms of a reduction of the maximum height of the domed surface. In some embodiments, doming of the permeabilisation surface is restricted by at least 95% in terms of a reduction of the maximum height of the domed surface.

[0039] In some embodiments, doming of the permeabilisation surface is restricted by at least 50% in terms of a reduction of the volume of the domed area. In some embodiments, doming of the permeabilisation surface is restricted by at least 60% in terms of a reduction of the volume of the domed area. In some embodiments, doming of the permeabilisation surface is restricted by at least 70% in terms of a reduction of the volume of the domed area. In some embodiments, doming of the permeabilisation surface is restricted by at least 80% in terms of a reduction of the volume of the domed area. In some embodiments, doming of the permeabilisation surface is restricted by at least 90% in terms of a reduction of the volume of the domed area. In some embodiments, doming of the permeabilisation surface is restricted by at least 95% in terms of a reduction of the volume of the domed area.

[0040] In some embodiments, doming of the permeabilisation surface is restricted by at least 50% in terms of a reduction in the surface area of the permeabilisation surface subjected to doming. In some embodiments, doming of the permeabilisation surface is restricted by at least 60% in terms of a reduction in the surface area of the permeabilisation surface subjected to doming. In some embodiments, doming of the permeabilisation surface is restricted by at least 70% in terms of a reduction in the surface area of the permeabilisation surface subjected to doming. In some embodiments, doming of the permeabilisation surface is restricted by at least 80% in terms of a reduction in the surface area of the permeabilisation surface subjected to doming. In some embodiments, doming of the permeabilisation surface is restricted by at least 90% in terms of a reduction in the surface area of the permeabilisation surface subjected to doming. In some embodiments, doming of the permeabilisation surface is restricted by at least 95% in terms of a reduction in the surface area of the permeabilisation surface subjected to doming.

[0041] The at least one protrusion is arranged to contact the majority of the permeabilisation surface to restrict doming of the permeabilisation surface. The term "majority", as used herein, is intended to mean over 50%. In some embodiments, the at least one protrusion is arranged to contact at least about 50% of the permeabilisation surface. In some embodiments, the at least one protrusion is arranged to contact at least about 60% of the permeabilisation surface. In some embodiments, the at least one protrusion is arranged to contact at least about 70% of the permeabilisation surface. In some embodiments, the at least one protrusion is arranged to contact at least about 75% of the permeabilisation surface. In some embodiments, the at least one protrusion is arranged to contact at least about 80% of the permeabilisation surface. In some embodiments, the at least one protrusion is arranged to contact at least about 85% of the permeabilisation surface. In some embodiments, the at least one protrusion is arranged to contact at least about 90% of the permeabilisation surface. In some embodiments, the at least one protrusion is arranged to contact at least about 92% of the permeabilisation surface. In some embodiments, the at least one protrusion is arranged to contact at least about 94% of the permeabilisation surface. In some embodiments, the at least one protrusion is arranged to contact at least about 95% of the permeabilisation surface.

[0042] In some embodiments, the at least one protrusion is arranged to contact a central area of the permeabilisation surface, wherein the central area is at least about 50% of the permeabilisation surface. The term "central area", as used herein, refers to a portion of the permeabilisation surface that includes the centre of the permeabilisation surface and extends continuously therefrom (i.e., there are no holes or gaps). In some embodiments, the at least one protrusion is arranged to contact a central area of the permeabilisation surface, wherein the central area is at least about 60% of the permeabilisation surface. In some embodiments, the at least one protrusion is arranged to contact a central area of the permeabilisation surface, wherein the central area is at least about 70% of the permeabilisation surface. In some embodiments, the at least one protrusion is arranged to contact a central area of the permeabilisation surface, wherein the central area is at least about 75% of the permeabilisation surface. In some embodiments, the at least one protrusion is arranged to contact a central area of the permeabilisation surface, wherein the central area is at least about 80% of the permeabilisation surface. In some embodiments, the at least one protrusion is arranged to contact a central area of the permeabilisation surface, wherein the central area is at least about 85% of the permeabilisation surface. In some embodiments, the at least one protrusion is arranged to contact a central area of the permeabilisation surface, wherein the central area is at least about 90% of the permeabilisation surface. In some embodiments, the at least one protrusion is arranged to contact a central area of the permeabilisation surface, wherein the central area is at least about 92% of the permeabilisation surface. In some embodiments, the at least one protrusion is arranged to contact a central area of the permeabilisation surface, wherein the central area is at least about 94% of the permeabilisation surface. In some embodiments, the at least one protrusion is arranged to contact a central area of the permeabilisation surface, wherein the central area is at least about 95% of the permeabilisation surface.

[0043] In some embodiments, in use the at least one protrusion contacts the permeabilisation surface to restrict doming of at least 50% of the permeabilisation surface. In some embodiments, in use the at least one protrusion contacts the permeabilisation surface to restrict doming of at least 60% of the permeabilisation surface. In some embodiments, in use the at least one protrusion contacts the permeabilisation surface to restrict doming of at least 70% of the permeabilisation surface. In some embodiments, in use the at least one protrusion contacts the permeabilisation surface to restrict doming of at least 75% of the permeabilisation surface. In some embodiments, in use the at least one protrusion contacts the permeabilisation surface to restrict doming of at least 80% of the permeabilisation surface. In some embodiments, in use the at least one protrusion contacts the permeabilisation surface to restrict doming of at least 85% of the permeabilisation surface. In some embodiments, in use the at least one protrusion contacts the permeabilisation surface to restrict doming of at least 90% of the permeabilisation surface. In some embodiments, in use the at least one protrusion contacts the permeabilisation surface to restrict doming of at least 95% of the permeabilisation surface. In some embodiments, in use the at least one protrusion contacts the permeabilisation surface to restrict doming of at least 96% of the permeabilisation surface. In some embodiments, in use the at least one protrusion contacts the permeabilisation surface to restrict doming of at least 97% of the permeabilisation surface. In some embodiments, in use the at least one protrusion contacts the permeabilisation surface to restrict doming of at least 98% of the permeabilisation surface. In some embodiments, in use the at least one protrusion contacts the permeabilisation surface to restrict doming of at least 99% of the permeabilisation surface. In some embodiments, in use the at least one protrusion contacts the permeabilisation surface to restrict doming of substantially all of the permeabilisation surface. In some embodiments, in use the at least one protrusion contacts the permeabilisation surface to restrict doming of 100% of the permeabilisation surface.

[0044] In some embodiments, the device is arranged such that at least 2 pl of ISF can be extracted from the permeabilisation surface. In some embodiments, the device is arranged such that at least 3 pl of ISF can be extracted from the permeabilisation surface. In some embodiments, the device is arranged such that at least 4 pl of ISF can be extracted from the permeabilisation surface. In some embodiments, the device is arranged such that at least 2 pl of ISF can be extracted from the permeabilisation surface over 10 minutes (i.e., 0.2 pl / min). In some embodiments, the device is arranged such that at least 3 pl of ISF can be extracted from the permeabilisation surface over 10 minutes (i.e., 0.3 pl / min). In some embodiments, the device is arranged such that at least 4 pl of ISF can be extracted from the permeabilisation surface over 10 minutes (i.e., 0.4 pl / min).

[0045] In some embodiments, the device is arranged such that from 2 pl to 20 pl of ISF can be extracted from the permeabilisation surface. In some embodiments, the device is arranged such that from 3 pl to 15 pl of ISF can be extracted from the permeabilisation surface. In some embodiments, the device is arranged such that from 4 pl to 10 pl of ISF can be extracted from the permeabilisation surface. In some embodiments, the device is arranged such that from 5 pl to 8 pl of ISF can be extracted from the permeabilisation surface.

[0046] In some embodiments, the device is arranged such that from 2 pl to 20 pl of ISF can be extracted from the permeabilisation surface over 10 minutes. In some embodiments, the device is arranged such that from 3 pl to 15 pl of ISF can be extracted from the permeabilisation surface over 10 minutes. In some embodiments, the device is arranged such that from 4 pl to 10 pl of ISF can be extracted from the permeabilisation surface over 10 minutes. In some embodiments, the device is arranged such that from 5 pl to 8 pl of ISF can be extracted from the permeabilisation surface over 10 minutes.

[0047] In some embodiments, the device is arranged such that at least 8 pl of ISF can be extracted per cm2of permeabilisation surface. In some embodiments, the device is arranged such that at least 9 pl of ISF can be extracted per cm2of permeabilisation surface. In some embodiments, the device is arranged such that at least 10 pl of ISF can be extracted per cm2of permeabilisation surface. In some embodiments, the device is arranged such that at least 11 pl of ISF can be extracted per cm2of permeabilisation surface. In some embodiments, the device is arranged such that at least 12 pl of ISF can be extracted per cm2of permeabilisation surface. In some embodiments, the device is arranged such that at least 13 pl of ISF can be extracted per cm2of permeabilisation surface. In some embodiments, the device is arranged such that at least 14 pl of ISF can be extracted per cm2of permeabilisation surface. In some embodiments, the device is arranged such that at least 15 pl of ISF can be extracted per cm2of permeabilisation surface. In some embodiments, the device is arranged such that at least 8 pl of ISF can be extracted per cm2of permeabilisation surface over 10 minutes (0.8 pl / cm2 / min). In some embodiments, the device is arranged such that at least 9 pl of ISF can be extracted per cm2of permeabilisation surface over 10 minutes (0.9 pl / cm2 / min). In some embodiments, the device is arranged such that at least 10 pl of ISF can be extracted per cm2of permeabilisation surface over 10 minutes (1 pl / cm2 / min). In some embodiments, the device is arranged such that at least 11 pl of ISF can be extracted per cm2of permeabilisation surface over 10 minutes (1.1 pl / cm2 / min). In some embodiments, the device is arranged such that at least 12 pl of ISF can be extracted per cm2of permeabilisation surface over 10 minutes (1.2 pl / cm2 / min). In some embodiments, the device is arranged such that at least 13 pl of ISF can be extracted per cm2of permeabilisation surface over 10 minutes (1.3 pl / cm2 / min). In some embodiments, the device is arranged such that at least 14 pl of ISF can be extracted per cm2of permeabilisation surface over 10 minutes (1.4 pl / cm2 / min). In some embodiments, the device is arranged such that at least 15 pl of ISF can be extracted per cm2of permeabilisation surface over 10 minutes (1.5 pl / cm2 / min).

[0048] In some embodiments, the device does not comprise any element that penetrates the skin tissue surface, such as needles or microneedles.

[0049] In some embodiments, the device is of unitary construction (i.e., one-piece construction). In some embodiments, the device comprises two parts (i.e., two- piece construction). In some embodiments, the device comprises two or more parts. In some embodiments, the device comprises three parts (i.e., three-piece construction). In some embodiments, the at least one protrusion is removable from the chamber.

[0050] In some embodiments, the device is constructed from resin, polyvinyl chloride (PVC), polypropylene, polyethylene, polystyrene, polyacrylamide, nylon, polyethylene terephthalate (PET), polyimide, polycarbonate (PC), acrylonitrile butadiene, polyether ether ketone (PEEK), polyurethane, polymethyl methacrylate, or PTFE (Teflon). In some embodiments, the device is constructed from a biocompatible material. In some embodiments, the device is 3D-printed. In some embodiments, the device is constructed from BioMed Clear resin or Flexible 81A resin (Formlabs).

[0051] In some embodiments, the device comprises an adhesive or adhesive layer for adhering the device to the skin tissue surface. This provides the advantage that the device is attached to the skin before the hypobaric seal is formed. In some embodiments, the device is for extracting one or more biomarkers. In particular embodiments, the one or more biomarkers are selected from the group consisting of L-tyrosine, stearic acid, urea, aminoguanidine, diphenylphosphinic acid, pyroglutamic acid, nitric acid, tryptophan, crotonyl isothiocyanate, urocanic acid, threonine, L-valine, myristic acid, cholest-5-ene, 3-methoxy-, (3. beta.)-, D-glucose, palmitic acid and betaine. In particular embodiments, the one or more biomarkers are selected from the group consisting of pyroglutamic acid, nitric acid, crotonyl isothiocyanate, threonine, L-valine, cholest-5-ene, 3-methoxy-, (3. beta.)-, and palmitic acid.

[0052] In some embodiments, the device comprises one or more sensors. The one or more sensors may be located at any suitable position of the device, for example in the chamber or on the surface of the one or more protrusions. Suitable sensors would be known to the skilled person and include, for example, electrochemical sensors, fluorescent sensors, colorimetric sensors, temperature sensors, pressure sensors and conductivity sensors. Such sensors could be used to analyse, for example, biomarkers and / or metabolites within the extracted ISF.

[0053] In some embodiments, the device is for delivering one or more agents to the skin tissue. In some embodiments, the device is for delivering one or more agents intradermally to the skin tissue. In some embodiments, the device is for delivering one or more agents subcutaneously. The skilled person will appreciate that any suitable agent may be for delivery to the skin tissue. In some embodiments, the one or more agents is a biomolecule. In some embodiments, the one or more agents is a biomolecule having a molecule weight of 5 kDa or more. In some embodiments, the one or more agents is a molecule or biomolecule having a molecular weight of 10 kDa or more. In some embodiments, the one or more agents is a molecule or biomolecule having a molecular weight of 20 kDa or more. In some embodiments, the one or more agents is a molecule or biomolecule having a molecular weight of 30 kDa or more. In some embodiments, the one or more agents is a molecule or biomolecule having a molecular weight of 40 kDa or more. In some embodiments, the one or more agents is a molecule or biomolecule having a molecular weight of 50 kDa or more. In some embodiments, the one or more agents is a molecule or biomolecule having a molecular weight of 70 kDa or more. In some embodiments, the one or more agents is a molecule or biomolecule having a molecular weight of 100 kDa or more. In some embodiments, the one or more agents is a molecule or biomolecule having a molecular weight of 150 kDa or more. According to a second aspect, there is provided a method of obtaining interstitial fluid from a skin tissue surface of a subject, wherein the method comprises: applying the skin permeabilisation device according to any preceding claim to a skin tissue surface of a subject; applying negative pressure to the chamber such that the chamber forms a hypobaric seal with the skin tissue surface and interstitial fluid is extracted from the permeabilisation surface into the chamber, wherein the at least one protrusion contacts a majority of the permeabilisation surface to restrict doming of the permeabilisation surface; and collecting interstitial fluid from the chamber.

[0054] The description above in relation to the skin permeabilisation device is equally applicable to this aspect.

[0055] The device is applied to the skin tissue surface in any suitable manner. Typically, the device is held in place against the tissue surface and until the hypobaric seal is formed. In some embodiments, the device is pressed against the skin tissue surface (i.e., pressure is applied to the device) for a period of time (e.g., about 1 minute). In some embodiments, the device is adhered to the skin tissue surface via an adhesive or adhesive layer.

[0056] Negative pressure is applied in any suitable manner such that the chamber forms a hypobaric seal with the skin tissue surface to provide a permeabilisation surface sealed within the chamber, and ISF is extracted from the permeabilisation surface.

[0057] In some embodiments, from -3.5 psi to -15 psi pressure is applied. In some embodiments, from -4 psi to -10 psi pressure is applied. In some embodiments, from -4.5 psi to -8 psi pressure is applied. In some embodiments, from -4.5 psi to -6 psi is applied. In some embodiments, about -4.5 psi pressure is applied.

[0058] In some embodiments, negative pressure is applied for 30 seconds to 180 minutes. In some embodiments, negative pressure is applied for 45 seconds to 90 minutes. In some embodiments, negative pressure is applied for 1 minute to 60 minutes. In some embodiments, negative pressure is applied for 2 minutes to

[0059] 45 minutes. In some embodiments, negative pressure is applied for 3 minutes to

[0060] 30 minutes. In some embodiments, negative pressure is applied for 4 minutes to

[0061] 20 minutes. In some embodiments, negative pressure is applied for 5 minutes to

[0062] 15 minutes. In some embodiments, negative pressure is applied for about 10 minutes.

[0063] Extracted ISF is collected from the chamber in any suitable manner. In some embodiments, ISF is collected via a sealable lid of the chamber by, for example, a pipette. In some embodiments, an absorbent substrate (e.g., gauze, pad or mesh, such as a honeycomb mesh) is present within the chamber and absorbs the extracted ISF. The method may therefore comprise depositing an absorbent substrate within the chamber prior to ISF extraction, and collecting the absorbent substrate from the chamber after ISF extraction. In some embodiments, in use, the absorbent substrate is positioned between the skin tissue surface and the at least one protrusion. In some embodiments, an extraction fluid (e.g., saline) is present within the chamber to receive the extracted ISF. The method may therefore comprise depositing an extraction fluid within the chamber prior to ISF extraction and collecting the extraction fluid from the chamber after ISF extraction.

[0064] The use of an absorbent mesh positioned within the chamber provides a further advantage of concentrating extracted ISF within the mesh. The inventors have developed a resin honeycomb mesh that is particularly effective, and may be used to concentrate ISF on the surface of a sensor integrated in the device (e.g., positioned on the surface of the at least one protrusion).

[0065] In particular embodiments, the method does not comprise penetrating the skin tissue surface with a foreign object (e.g. a needle, microneedle or bore).

[0066] According to a third aspect, there is provided a method of delivering an agent to a skin tissue of a subject, wherein the method comprises: applying the skin permeabilisation device according to any preceding claim to a skin tissue surface of a subject; introducing an agent for delivery to the skin tissue into the chamber; applying negative pressure to the chamber such that the chamber forms a hypobaric seal with the skin tissue surface and the agent is delivered through the permeabilisation surface into the skin tissue, wherein the at least one protrusion contacts a majority of the permeabilisation surface to restrict doming of the permeabilisation surface.

[0067] In some embodiments, the agent is a biomolecule. In some embodiments, the agent is a biomolecule having a molecule weight of 5 kDa or more. In some embodiments, the agent is a molecule or biomolecule having a molecular weight of 10 kDa or more.

[0068] In some embodiments, the agent is introduced into the chamber through a sealable lid or port after the device is applied to the skin tissue surface.

[0069] In some embodiments, the agent is introduced into a reservoir of the at least one protrusion.

[0070] In some embodiments, from 50 to 2000 pl agent is introduced into the chamber. In some embodiments, from 100 to 1000 pl agent is introduced into the chamber. In some embodiments, from 150 to 750 pl agent is introduced into the chamber. In some embodiments, from 200 to 500 pl agent is introduced into the chamber.

[0071] The description above in relation to the skin permeabilisation device and the method of obtaining interstitial fluid from a skin tissue surface of a subject is equally applicable to this aspect.

[0072] A skilled person will appreciate that all aspects of the invention, whether they relate to, for example, the skin permeabilisation device, are equally applicable to all other aspects of the invention. In particular, aspects of the skin permeabilisation device, for example, may have been described in greater detail than in other aspects of the invention, for example, the method of obtaining interstitial fluid. However, the skilled person will appreciate where more detailed information has been given for a particular aspect of the invention, this information is generally equally applicable to other aspects of the invention.

[0073] All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety.

[0074] Brief Description of the Drawings

[0075] The invention will now be described in detail, by way of example only, with reference to the figures.

[0076] Figure 1: A schematic illustrating the means by which tissue stretching allows extraction of chemicals directly from the skin without damage.

[0077] Figure 2: Two examples of devices without a loading post.

[0078] Figure 3: A schematic showing the concept of the design for animal studies.

[0079] Figure 4: Process of extracting human ISF using hypobaric pressure: (a) application of pressure for 1 minute; (b) assembling and attaching of device on dorsal forearm; (c) pipetting 150pL of extraction buffer; d) application of hypobaric pressure for 5-10 minutes; (e) collection of ISF and extraction buffer to be frozen until analysis.

[0080] Figure 5: A schematic showing (top) the one-part device with a liquid collection (absorbent) layer which is pre-wetted with a liquid prior to application to the skin (bottom) envisaged application process for the device with a baby during feeding.

[0081] Figure 6: Macromolecule extraction efficiency across ex vivo rat skin under the application of different hypobaric pressures. Below a pressure of -4.5 psi the extraction was molecular weight dependent, suggesting a diffusion-based process, while at pressure of -4.5 and above the extraction was molecular weight independent, suggesting convective transport.

[0082] Figure 7: (a) Confocal microscopy imaging of rat skin (20 mm) after a 1 h extraction of dextran 10 KDa and 150 kDa under atmospheric (-) and -4.5 psi hypobaric pressure conditions (+), at magnification X100 and showing the hair follicle area (i and iii) and the basement membrane, epidermis and stratum corneum area (ii and iv), at magnification X100. Blue: DAPI, green; Dextran FItC. b) The change in diameter of skin appendages under hypobaric pressure and c) cross-section confocal image of a skin appendage using confocal microscope (X10) showing effect of pressure on diameter.

[0083] Figure 8: (a) ISF extraction across rat paw skin using hypobaric pressure in the presence of normal ISF flow and immediately after the cessation of the circulation and ISF flow (n = 5 ± SD). (b) Extraction of rat TNF-alpha across full thickness dorsal mice skin, rat skin, porcine ear skin and dermatomed (1 pm) human skin ex vivo under atmospheric (-) or under -4.5 psi hypobaric pressure (+) for 1 h (n = 5 ± SD).

[0084] Figure 9: Left, quantification of cytokines expressed in acute high-grade inflammation in skin biopsy and ISF samples. Right, TNFa in living and culled rats, with and without hypobaric pressure.

[0085] Figure 10: Non-invasive ISF profiling detects changes in cytokines during low-grade skin inflammation. (A) Rat dorsal skin appearance immediately after repeated skin stretching for longitudinal ISF extraction. (B) H&E staining of rat skin after the application of repeated skin stretching (at 0, 3, 6 and 24 h) on healthy skin (control) and on skin with ED. (C) Cartoon of ED formation. (D) Transepidermal water loss (TEWL) across rat skin measured following a single skin stretching application (-4.5 psi, 10 min) on heathy skin (control) and on skin with ED (n = 3 ± SD). (E) Changes in TEWL across rat skin over 24 h following ED induction (n = 3 ± SD). (F) Total protein levels extracted across healthy control skin and skin with ED following repeated ISF extraction via skin stretching over 24 post the induction of ED (n = 6 ± SD). (G) ISF inflammatory cytokines extracted across the skin, via skin stretching, post ED induction (n = 6 ± SD). (H) Tissue inflammatory cytokines post-ED induction detected using tissue explant (n = 6 ± SD).

[0086] Figure 11: Multiplex Cytokine data analysis of ISF from melanoma bearing mice. Longitudinal sampling of ISF pre and post tumour inoculation highlighting the presence of 6 spiked cytokines, results are expressed as mean ±SD, n = 3-5. * Represents the significant difference from baseline levels where p < 0.05. Figure 12: Multiplex Cytokine data analysis of ISF, Serum and Tumour lysate for melanoma bearing mice. (A) Venn diagram showing the 3 biofluids analysed after subtraction of cytokines present in background healthy fluids. (B) Heat map to determine the fold changes in cytokine levels post tumour inoculation in ISF, serum, and tumour lysate compared to baseline levels. Values below LOD are referred to as zero on heatmap.

[0087] Figure 13: LCMS proteomic data analysis of ISF, Serum and Tumour lysate for melanoma bearing mice (A) Heat map to visualise the proteomic profile of ISF post tumour implantation on different time points. (B) Heat map to visualise the proteomic profile of ISF VS Serum post tumour implantation on the endpoint of the study.

[0088] Figure 14: (A) Volume of ISF extracted (pL) from healthy human volunteers' forearm volunteers when using 3 different device sizes. (B) Volume of ISF extracted (pL) from healthy per cm2of skin human volunteers forearm volunteers when using 3 different device sizes application. Results are expressed as mean ±SD, n = 3.

[0089] Figure 15: Left panel. Volume of ISF extracted (pL) from healthy human volunteers' forearm using different extraction times. Right panel. Volume of ISF extracted (pL) from healthy human volunteers' forearm when repeated on the same site. Results are expressed as mean ±SD, n = 3.

[0090] Figure 16: Volume of ISF extracted (pL) from healthy human volunteers' forearm using the 1-part device in two different orientations that are possible as it does not use an extraction buffer. Results are expressed as mean ±SD, n = 3.

[0091] Figure 17: Non-invasive extraction via skin stretching detect unique biomarkers for induced epidermal dysfunction. (A) Cartoon shows the skin structure and detectable cytokines for healthy skin and skin with epidermal dysfunction (B) Human skin images (Healthy skin, immediately after 10 min Hypobaric pressure, 30 min after 10 min Hypobaric pressure, 24 hours after 10 min Hypobaric pressure, (Human skin with ED, immediately after 10 min

[0092] Hypobaric pressure, 30 min after 10 min Hypobaric pressure, 24 hours after 10 min Hypobaric pressure) (C,D,E) Levels of IL-1B, IL-6 and TNF-alpha levels in pre- and post-ED induction, results are expressed as pg / mL per pL of ISF. (F) Heatmap showing the fold change in proteins associated with natural skin inflammation, results are expressed as fold change. (G) structure of myosin found to be a key protein in the proteomics.

[0093] Figure 18: Non-invasive ISF sampling can detect inflammaging biomarkers in healthy skin. (A) Cartoon depicting the changes to the skin that occur with aging resulting in low grade inflammation. (B) TEWL values of different age groups. (C) Elasticity values of different age groups. (D) Levels of IL-1B in different age groups, results are expressed as pg / mL per pl of ISF. (E) Heatmap showing the fold change in proteins associated with natural skin inflammaging in different age groups, results are expressed as fold change. (F) Cartoon showing the aging phenotype in the skin.

[0094] Figure 19: Top left image of the change in skin stretch upon use of the loading post. Top right image of human skin after use of the loading post. Bottom left volume of ISF extracted (pL) from healthy human volunteers' forearm using different extraction conditions. Results are expressed as mean ±SD, n = 3. Bottom right. A drawing of the device with two different types of loading posts.

[0095] Figure 20: Left: volume of ISF extracted (pL) from healthy human volunteers' forearm using different extraction conditions. Results are expressed as mean ±SD, n = 3. Right: Drawings of the device with no loading post (Standard) or different types of loading posts (12mm, 15mm, 17mm, concentric).

[0096] Figure 21: A list of the chemicals that were extracted in the ISF using three different devices (2-part device without applying vacuum (ATM), 2-part device when applying vacuum (HYP), and the 2-part device with a loading post and applying vacuum (HYP-loading post). Results are expressed as mean of n = 3.

[0097] Figure 22: Data to show the extraction of 9 different cytokines from the skin of healthy adults using the 2-part ISF collection device. Data is mean ± one standard deviation (n=6).

[0098] Figure 23: Tolerability profiles of various 3D printing materials used in device application over time. The area under the curve (AUC) represents the patient-reported pain scores, with lower values indicating less discomfort. Flexible 80A is shown in black, BioMed Clear 65% in pink, BioMed Clear 80% in purple, and BioMed Clear 100% in blue. Data points at 5 and 10 minutes application times show that Flexible 80A consistently results in the lowest discomfort levels. Statistical significance is denoted by asterisks (*p < 0.05, **p < 0.01, ***p < 0.001), with the number of asterisks indicating the level of significance between comparisons. Error bars represent standard deviation, highlighting the variability within each condition. Error bars indicate standard deviation, underscoring the variability within each group, n=6 for each condition.

[0099] Figure 24: Design of three exemplary devices that can be used to extract ISF from animals, which have a smaller surface area compared to humans and thus need a different device design. Figure 25: Tolerability of the 3-part animal device with loading post.

[0100] Figure 26: Efficacy of the 3-part animal device with and without the loading post in extracting the skin ISF. Data is mean ± SD, n=3.

[0101] Figure 27: The extraction of ISF using the 2-part device that generates a skin dome and the loading post device using different extraction times (n=3 extractions).

[0102] Figure 28: An example of a honeycomb collection mesh to collect ISF from the loading post device. (A) The mesh design with exemplar dimensions. (B) The use of the mesh to collection ISF during skin stretching using the loading post device.

[0103] Figure 29: The extraction of ISF using the 2 part device with a loading post, the loading post with a surgical gauze and the loading post with the honeycomb mesh (n=3-7 extractions).

[0104] Figure 30: Different one-part device configurations used with the rhodamine dye to understand the contact of the device with the skin and the penetration of the dye.

[0105] Figure 31: Delivery of rhodamine dye into porcine skin. (A) The images of the skin after the application of different variations of the one-part device. (B) Amount of rhodamine delivered into the skin (n=l). Modification of the loading post device with the addition of a small reservoir or the addition of a gauze or sponge soaked in the rhodamine provided enhanced delivery over the atmospheric conditions or the device without a loading post.

[0106] Figure 32: Comparison of the delivery of 150 KDa dextran into porcine skin using the 2-part device without a loading post, the 1 part loading post device with a gauze attached to the loading post, the 1 part loading post device with a reservoir. Data is mean ± 1 standard deviation (n=3).

[0107] Detailed Description

[0108] An innovative device that is capable of permeabilising the skin without the use of a needle to allow both the delivery and extraction of chemicals without damaging the tissue has now been designed and tested to solve the problems associated with needleless drug delivery and biomarker extraction. The following examples will show how the device has been used in ex vivo, in vivo animal and human studies. The principle of the device is that it is applied to the skin, a vacuum is applied, the skin is then stretched, but it is not allowed to form a dome and this opens the hair follicles and spaces between the cells are enlarged and a pressure gradient is set up to draw molecules out of the tissue and let molecules penetrate into it (Figure 1). Both large >1 kDa and small molecules can be exchanged across the tissue in a process that is independent of molecular weight, but it appears to be particularly efficient at drawing out large biological agents with a molecular weight of >10kDa (Figure 1).

[0109] It has been surprisingly demonstrated that if a "post" is inserted into the centre of the device, dome formation is suppressed and the skin stretched laterally, such that up to 4 pL of ISF can be sampled from the skin (equivalent to microneedles, which is an invasive method that physically pierces the tissue (2)). As skin stretching allows repeated sampling or delivery into the same skin site, and it can be applied to disease tissue, this device has the potential to be used in a wide range of medical applications. Supporting data are provided in the following examples. on chemical extraction from the

[0110] Methods

[0111] Rat skin was obtained by Schedule 1 killing of the rats by intraperitoneal injection of sodium phenobarbital. The dorsal hair was removed using an animal hair clipper (Wella Professionals, Jarosov, Czech Republic). The excess fat adhering to the dermis side was removed carefully using a scalpel. The rat skin, with thickness 1.15 ± 0.05 mm, as measured by digital calliper (SLS, Nottingham, UK) harvested from animals was cut into pieces of suitable size (3 x 3 cm).

[0112] The Franz cell glass receptor compartment was filled with 500 pg / mL of each of the following: 100 pg / mL purified FITC-Dextran 10 kDa, 100 pg / mL purified FITC-Dextran 150 kDa (all commercial dextran's supplied by Sigma- Aldrich, Dorset, UK), and 350 pg / mL TNF-alpha 17 kDa (Merk Life Sciences Limited, Gillingham, UK). A 3D printed extraction device, without a loading post, was attached to the Franz cell receptor (printed using a Form 3, 3D printer, Formlabs, Massachusetts, USA) under different hypobaric pressures (-2.8 psi, - 4.5 psi or -7.9 psi) for 1 h in water bath maintained at 37 °C. After 1 h, hypobaric pressure was removed, and the fluid was collected from the 3D printed stretching device for analysis.

[0113] The extracted fluid was analysed for the dextran and the TNF-alpha. The FITC-Dextrans were quantified using a multimode microplate reader (Tecan Spark®, Reading, UK) at an excitation wavelength of 485 nm, fluorescent emission wavelength of 535 nm, manual gain of 38 that was fixed for every plate. The Rat TNF-alpha was measured using an ELISA kit coated with capture antibodies (Invitrogen, Thermofisher Scientific, UK) by a multimode microplate reader (Tecan Spark ®, Reading, UK). The effect of skin stretching on the extraction of FITC-Dextrans and TNF-alpha was presented as extraction efficiency (%)■

[0114] Results

[0115] Using a skin stretching hypobaric pressure of 4.5 psi or above extracted molecules out in a molecular weight independent manner suggesting convective transport, i.e., movement through pores generated using skin stretching (Figure 6). Below a pressure of -4.5 psi the extraction was molecular weight dependent suggesting that it followed a diffusion-based process.

[0116] Example 2 - Pathways of chemical extraction using stretching

[0117] Methods

[0118] Ex vivo extraction of the purified FITC-dextrans was conducted across excised dorsal rat skin (S-D male rats, 6-8 weeks old), which was used within three months of acquisition. The rat skin was prepared by the removal of subcutaneous tissue and hair. The prepared full thickness skin (thickness 0.98 ± 0.18 mm) was mounted onto individually calibrated upright Franz diffusion cells (Southampton University, UK) (diffusional area of ~2.1 cm2and receptor volume of ~10.8 ml) using the 3-D printed chamber (printed using a Form 3, 3D printer, Formlabs, Massachusetts, USA), without a loading post, as the donor compartment. The receiver compartment was filled with PBS (pH 7.4). Assembled Franz cells were placed onto a Variomag® magnetic stirrer plate immersed in a water bath (Grant Instruments, UK) at 37°C to maintain surface of the skin at 32°C for a 30 min equilibration period. After equilibriation, the PBS was removed and the receptor compartment was filled with 100 pg / ml solutions of either the purified 10 KDa, 70 KDa or 150 KDa FITC- dextran in PBS (pH 7.4) (supplied by Sigma-Aldrich, Dorset, UK). A 1 ml aliquot of PBS was introduced in the donor compartment Franz cells replaced onto the water bath. The studies were conducted for a duration of 1 h under either atmospheric pressure conditions or at -4.5 PSI hypobaric pressure generated and maintained using a calibrated hand pump.

[0119] After the 1 h extraction time, the cells were removed, the pressure discontinued and the solution inside the donor compartment was carefully collected, and the volume was measured and recorded. Aliquots from the donor solution were analysed immediately for FITC content using a using a Tecan Spark plate reader (Tecan Ltd, UK) at excitation wavelength of 485 ± 20 nm and emission wavelength of 535 ± 20 nm (assays previously validated and found fit for purpose). All the extraction experiments were repeated 4-5 times.

[0120] At the end of the ex vivo extraction study (1 h under controlled stretching), the skin was removed from the Franz cells, any remaining formulation on the surface of the skin was carefully removed by washing the skin five times using DI water and cotton buds, and the application of two tape strips. The skin was cut into strips (three from each Franz cell), embedded in OCT and frozen gently using chilled isopentane (isopentane was chilled to near freezing by immersing the isopentane container in liquid nitrogen) then cut into lateral tissue sections with a thickness of 20 pm using a cryostat (Bright Instruments, Huntingdon, UK). The tissue sections were mounted onto microscopy slides (SuperFrost, Fisher Scientific), washed in PBS (1 min) and fixed in 10% formalin (10 s) then mounted using SlowFade Gold Antifade Mountant, with DAPI, and sealed with a cover slip. Confocal microscopic images were obtained with Al inverted confocal with spectral detector by Nikon (Kingston Upon Thames, UK) at a magnification of 20X (air objective) and 100X (Oil obj). Images were obtained with two fluorescence channels: DAPI (405 nm) and FITC (488 nm). Images were analysed using NIS-Elements Imaging Software (Nikon).

[0121] A StageFlexer™ (Flexcell International, Burlington, USA) was used to view the real-time skin stretching under hypobaric pressure (from -3 psi to - 8 psi). The StageFlexer™ was prepared by inserting the loading station with freshly prepared rat skin, stained with DAPI, on top of one another and securing the assembly using the retaining screws. The cell was mounted with a glass coverslip. The StageFlexer™ was connected to a handheld vacuum pump with a gauge (Fischer Scientific, Leicester, UK), which allowed the application of a hypobaric pressure in an identical manner to the ex vivo skin permeation studies. Confocal microscopic images were obtained with Al inverted confocal with spectral detector by Nikon (Kingston Upon Thames, UK) at a magnification of 10X. Images were obtained with two fluorescence channels: DAPI (405 nm) and FITC (488 nm). Images were analysed using NIS-Elements Imaging Software (Nikon).

[0122] Results

[0123] Chemical extraction when stretching the skin occurs both through the spaces between cells and through the hair follicles, but the hair follicle pathway predominates for large molecules (Figure 7a). There was an increase in hair follicle opening as more stretching pressure was applied (Figure 7b) and the stretching opened the hair follicles (Figure 7c).

[0124] 3 - Chemical extraction across different tvoes of skin

[0125] Methods

[0126] Ex vivo extraction of rat TNF-alpha was conducted across excised dorsal rat skin, mouse skin, porcine skin and human skin. The skin was prepared by the removal of subcutaneous tissue and hair. The prepared full thickness skin was mounted onto individually calibrated upright Franz diffusion cells (Southampton University, UK) (diffusional area of ~2.1 cm2and receptor volume of ~10.8 ml) using the 3-D printed chamber, without a loading post, as the donor compartment to stretch the skin (printed using a Form 3, 3D printer, Formlabs, Massachusetts, USA). The receiver compartment was filled with PBS (pH 7.4). Assembled Franz cells were placed onto a Variomag® magnetic stirrer plate immersed in a water bath (Grant Instruments, UK) at 37°C to maintain surface of the skin at 32°C for a 30 min equilibration period. After equilibration, the PBS was removed and the receptor compartment was filled with solutions of TNF-alpha (Merk Life Sciences Limited, Gillingham, UK). A 1 ml aliquot of PBS was introduced in the donor compartment Franz cells replaced onto the water bath. The studies were conducted for a duration of 1 h under either atmospheric pressure conditions or at -4.5 PSI hypobaric pressure generated and maintained using a calibrated hand pump.

[0127] After the 1 h extraction time, the cells were removed, the pressure discontinued and the solution inside the donor compartment was carefully collected, and the volume was measured and recorded. Aliquots from the donor solution were analysed immediately for TNF-alpha analysis using an ELISA assay as per the manufacturer's instructions (Invitrogen, Thermofisher Scientific, UK). All the extraction experiments were repeated 4-5 times.

[0128] For the in vivo rat studies all procedures were conducted as per the UK Animal Scientific Procedures Act (1986) and Amendments Regulations (2012) and approved by the King's College London Animal Care and Ethics Committee. Sprague Dawley (S-D) rats (7-9 w / o, male, ca. 220-250 g; Charles River, Kent, UK) were caged in groups of 4 with free access to water and food. A temperature of 19-22 °C was maintained, with a relative humidity of 45-65%, and a 12 h light / dark cycle. Animals were acclimatized for 7 days before each experiment. All animals were culled by a schedule 1 method upon the termination of the experiment. The extraction device was a glass chamber, without a loading post. It was applied to the rat skin for 20 min during animal anaesthesia or after culling the animals to assess the effect of the systemic circulation on the ISF flow on the extraction.

[0129] Results

[0130] The extractions in the anaesthetised rats (with ISF flow) vs the culled rats (no ISF flow) showed that when the circulation does not push the ISF around the body it cannot be extracted, but in living animals ISF can be extracted from the tissue (Figure 8). Interestingly, the ex-vivo comparisons across different types of tissue showed that stretching works more effectively in human skin compared to some of the animal skins presumably due to the elastic properties of the tissue, this is surprising as the permeability of human skin is generally less than animal skins (Figure 8).

[0131] Examole 4 - Longitudinal extraction of chemical from the skin of animals in vivo

[0132] Methods

[0133] All procedures were conducted as per the UK Animal Scientific Procedures Act (1986) and Amendments Regulations (2012) and approved by the King's College London Animal Care and Ethics Committee. Sprague Dawley (S-D) rats (7-9 w / o, male, ca. 220-250 g; Charles River, Kent, UK) were caged in groups of 4 with free access to water and food. Female C57BL6 mice (6- 9 weeks old, ca. 18-25 g; Charles River, Kent, UK) were caged in groups of 3 with free access to water and food. A temperature of 19-22 °C was maintained, with a relative humidity of 45-65%, and a 12 h light / dark cycle. Animals were acclimatized for 7 days before each experiment. All animals were culled by a schedule 1 method upon the termination of the experiment, humidity of 45 - 65%, and a 12 h light / dark cycle. Animals were acclimatised for 7 days before each experiment.

[0134] Chemical extraction using skin stretching during acute inflammation

[0135] Acute local inflammation was induced in rat paws (S-D, male, 7-8 w / o, 270-300 g) by the intra-planter injection of 0.1 ml of 1% carrageenan (Cg) solution. At 4 h post-injection, the the stretching device, without a loading post, was placed onto the non-glaborous skin of the inflamed paw and on the untreated contralateral paw (control) and 0.3 ml of PBS (pH 7.4) was introduced into the stretching device. The chemical extraction was initiated by the induction of the hypobaric pressure (-4.5 psi, 20 min) into the stretching device. At the end of the study, a biopsy was also taken from each tested paw. The level of cytokines within the tissue was determined by the explant method (Sopasakis et al., 2005; Thalmann et al., 2008). To achieve this, the skin samples were incubated individually in a 24-well plate and 0.5 ml of cell culture media (Gibco™ DMEM, high glucose, pyruvate cell culture medium, Fisher Scientific, UK) for 24 h in a CO2 incubator at 32°C. The extraction samples and the cell culture medium explant samples were stored at -80°C until analysis for the pro-inflammatory cytokines TNF-alpha, IL-l-beta and IL-6 using coated ELISA immunoassay as per the manufacturer's instructions (Invitrogen, UK). All the extraction experiments were repeated five times. The physical changes in the inflamed paw size were determined by triplicate measurements carried out at 1 h intervals up to 5 h after the Cg injection. The results were expressed as paw swelling % (PS).

[0136] Chemical extraction using skin stretching during low-grade inflammation (Epidermal Dysfunction)

[0137] Low-grade inflammation was generated using the epidermal dysfunction (ED) model in rats (S-D, male, 7-8 w / o, 270-300 g). The animals' lower dorsal skin was shaved with clippers and both flanks were used; the left for the ED model and the right for the untreated control. The acute epidermal dysfunction was induced on a 2 x 2 cm area by the consecutive application of tape strips (Scotch 845 book tape, 3 M, UK) to obtain a 6 to 7 -fold increase in transepidermal water loss (TEWL) (AquaFlux, Bioxx, UK), chemical extraction using skin stretching was completed at the ED site and the untreated site using a 3D printed device without a loading post under a hypobaric pressure of -4.5 psi maintained for 10 min prior to the ED induction and at 0, 3 h, 6 h and 24 h post- ED induction. The skin was inspected visually after the extraction. The extraction solutions were stored at -80 °C until analysis for concentration for TNF-alpha, IL- Ibeta and IL-6 using coated ELISA immunoassay. All the extraction experiments were repeated 5 times. The ELISA assay was conducted as per steps described in the assay's protocol (Invitrogen, UK). To determine the effect of skin stretching on the integrity of the skin, skin biopsies were obtained from the inflamed and control paw with and without skin stretching (see histology studies section for details).

[0138] Skin biopsies were obtained from untreated healthy paw, untreated inflamed paw, at 6 h post-intraplantar injection of the 1% carrageenan, and from the paw of animals challenged under the same hypobaric pressure conditions employed in the anti-inflammatory assay studies. Skin biopsies were also obtained from healthy dorsal skin and dorsal skin with epidermal dysfunction (24 h post-induction) prior and after the application of skin starching at predetermined time points. The skin samples were fixed with 4 % neutral- buffered formalin for 24 h at 4° C, then were cut in strips, embedded in O.C.T. and frozen as described for the confocal imaging section. Cross-section slices of 10 pm thickness were obtained using a Bright Model OTF cryostat (Bright Instruments, Huntingdon, UK). The samples were then stained following the Ellis Haematoxylin and Eosin (H&E) staining protocol (ref) and dehydrated with different volumes of ethanol (75%, 95%, and 100%) and xylene, before being mounted in DPX and covered with glass cover slips. The samples were analysed using a Leica DM 200 Led light microscope (Leica Microsystems, Wetzlar, Germany) equipped with a Leica digital camera (Model DFC 295) at a magnification of 20x. Images were processed using Las v4.4 Imaging Software (Leica Microsystems, Wetzlar, Germany).

[0139] Mouse Melanoma Study

[0140] To longitudinally sample from mice implanted with B16F10 melanoma cells to form a melanoma model, mice were anaesthetised by inhalation of (1-2%) isoflurane I (1-2 %) O2 and placed on a heating mat (Harvard Apparatus, Cambridge, UK) in the ventral position maintained at 37 °C throughout the experiments. The dorsal hair was carefully removed (to avoid any damage to the skin) with an animal hair clipper (Centura, London, UK). The mice were injected with the B16F10 cells (1 x 106harvested cells) subcutaneously into the right and left rear flanks of the female C57BL6 mice (n = 12, 6-9 weeks old), i.e., bilateral inoculation. Dual extractions directly from the tumour sites were performed on days 1, 2, 3, 4, 7, 8, 9, and 10 post tumour inoculation. The 3D printed stretching devices without a loading post was placed on the lower flank of the mice, and 150 pL sterile PBS (pH 7.4) was introduced into the device via the top of the tubular structure, making direct contact with the mouse skin. A pressure of -4.5 psi was applied for 20 min to the skin site via an in-house developed automated pump that self-corrected in case of pressure loss. After 20 min, pressure was released, and PBS in contact with the skin was carefully collected using a pipette. The skin area was monitored for any persistent redness within 24 hours, and the tumours were observed to ensure the device did not affect their structure. Blood samples were collected on days 7 via tail vein and on day 10 by cardiac puncture post mice cull. Blood samples were left to clot at room temperature for 2 hours, followed by centrifugation at 5,000 rpm for 10 minutes (Biofuge, Heraeus, Germany), and supernatant was collected as serum. Mice were culled by schedule 1 method upon experiment termination, and tumours and surrounding skin biopsies were collected. The same procedure was repeated for control healthy, tumour-free mice (n=6, 6-9 weeks old), and ISF, serum and skin biopsies were collected. Tumour biopsies were also taken and lysed in a lysis buffer consisting of Tris Buffered Saline (pH 8.0), Sodium Chloride, Ethylenediaminetetraacetic acid (EDTA), and Nonidet P-40, all obtained from Sigma-Aldrich® (Dorset, UK). The lysis buffer was prepared by adding 800 mg of NaCI and 58.4 mg of EDTA to 99 mL of TRIS buffered saline. After 1 hour of dissolution for EDTA, 1 mL of Nonidet P-40 was added. Tumour biopsies (average weight of 500 mg) were chopped and incubated in glass vials with 5 mL of prepared lysis buffer overnight at 4°C. The next day, solutions were homogenised using a tissue homogeniser (Ultra Turrax, Thermofisher, Leicester, UK) until no tissue remained intact. Samples were centrifuged for 15 minutes at 13,000 rpm (Biofuge, Heraeus, Germany), and the supernatant was collected and filtered with 0.45 pm membrane filter. The collected ISF, serum and tumour lysates were stored at -80°C until further analysis. Prior to sample analysis, ISF and serum were treated as neat samples without preparation. ISF, serum and tumour lysate samples underwent analysis for total protein content, multiple cytokines and proteomic analysis.

[0141] LCMS proteomic analysis

[0142] Chromatographic separation was performed using a U3000 UHPLC NanoLC system (Thermofisher Scientific, UK,). Peptides were resolved by reversed phase chromatography on a 75 pm C18 Pepmap column (50 cm length) using a three- step linear gradient of 80% acetonitrile in 0.1% formic acid. The gradient was delivered to elute the peptides at a flow rate of 250 nL / min over 60 minutes starting at 5% B (0-5 minutes) and increasing solvent to 40% B (5-40 minutes) prior to a wash step at 99% B (40-45 minutes) followed by an equilibration step at 5% B (45-60 minutes).

[0143] The eluate was ionised by electrospray ionisation using an Orbitrap Fusion Lumos (Thermofisher Scientific, UK) operating under Xcalibur v4.3. The instrument was first programmed to acquire using an Orbitrap-Ion Trap method by defining a 3s cycle time between a full MS scan and MS / MS fragmentation by collision induced dissociation. Orbitrap spectra (FTMS1) were collected at a resolution of 120,000 over a scan range of m / z 375-1600 with an automatic gain control (AGO) setting of 4.0 x 105(100%) with a maximum injection time of 35 ms. Monoisotopic precursor ions were filtered using charge state (+2 to +7) with an intensity threshold set between 5.0 x 103to 1.0 x IO20and a dynamic exclusion window of 35 s ± 10 ppm. MS2 precursor ions were isolated in the quadrupole set to a mass width filter of 1.6 m / z. Ion trap fragmentation spectra (ITMS2) were collected with an AGC target setting of 1* 104(100%) with a maximum injection time of 35 ms with CID collision energy set at 35%.

[0144] Raw mass spectrometry data were processed into peak list files using Proteome Discoverer (ThermoScientific; v2.5). The raw data file was processed and searched using the Sequest (9) search algorithm against the Uniprot Mouse Taxonomy database (37,517 entries; March 2023). Database searching was performed at a stringency of 1% FDR including a decoy search with precursor ion intensity quantification.

[0145] All samples were resuspended in 12 pL of resuspension buffer (2% ACN in 0.05% FA) to a normalised concentration of 0.42 g / pL, 6pL (2.5 mg on column) was injected to be analysed by LC-MS / MS. All proteomic LCMS sample preparation and analysis were performed at KCL Denmark Hill Proteomics Facility. Data was generated in Scaffold (version 4.8.5, Portland, USA) and protein identifications were reported at a threshold of 99.0%, with a minimum of three peptides assigned per protein, at a peptide threshold of 95%.

[0146] Multiplex cytokine analysis

[0147] To measure the cytokines, a V-Plex Proinflammatory Panel 1 multiplex kit and V-Plex Cytokine Panel 1 multiplex kit (Mesoscale Diagnostics, Maryland, USA) were utilised. These two kits enabled the simultaneous analysis of 19 cytokines, including IFN-y, IL-lg, IL-2, IL-4, IL-6, IL-5, IL-10, IL-12p70, KC-GRO, TNFo, IL- 9, MCP-1, IL-33, IL-27p28, IL-15, IL-17A, MIP-l-o, IP-10, and MIP-2. The test was performed as per the MSD kit instructions.

[0148] Results

[0149] The injection of carrageenan acts as the inflammatory stimulus, leading to the activation of macrophages and subsequent cytokine release and inflammation (Figure 9). A time-dependent increase in paw volume after the carrageenan injection was observed. The maximal oedema, a 120% increase from baseline, was reached at 5 h post injection. In the anaesthetised animals, i.e., those with blood and ISF flow the recovery of TNFo from the tissue was significantly higher, with a near 5-fold increase compared to the culled animals, i.e., without blood and ISF flow. This suggests that stretching in the live animals assisted ISF sampling. Analysis of the fluid extracted from the rat paw using the skin stretching device revealed similar cytokine concentrations compared to traditional skin biopsy methods to the ISF sampling technique (Figure 9). The ability to non- invasively and effectively quantify cytokine profiles in ISF underscores the potential of this technique to advance our understanding of the acute inflammatory process and, possibly, its resolution.

[0150] Epidermal dysfunction (ED) model was induced in healthy adult rats to mimic low-grade inflammation that occurs during skin aging (Hu et al., 2017). Epidermal dysfunction in aged skin leads to the release of pro-inflammatory cytokines release (including TNF-a, IL-la, IL-lb, IL-6 and IL-7) which contributes to the state of skin inflammaging (Figure 10C). ED was induced by the partial removal of the stratum corneum (SC) (Figure 10B) and a 3.8-fold increase in TEWL compared to healthy skin (Figure 10E). ISF sampling using the skin stretching device enable repeated ISF sampling in a non-invasive manner in healthy and ED skin as there was no visible damage to the skin (Figure 10A) and no changes in the skin histology (Figure 10B). Skin stretching resulted in temporary permeabilisation of skin barrier as evidenced in elevated TEWL immediately post stretching. These changes in skin permeabilsation were reversible in both healthy and ED skin as TEWL returned to baseline after approximately 15 minutes after stretching was removed (Figure 10D). Cytokines TNF-a, IL-lb and IL-6 were detected in the ISF extracted using skin stretching (Figure 10G). When the skin samples were surgically removed and the three cytokines directly extracted from the tissue, the profile obtained was consistent with the ISF sampling approach (Figure 10H). As ED resulted in partial removal of the SC, this could have facilitated ISF and cytokine extraction. However, the determination of the total protein levels in ISF extracted from ED skin and healthy skin remain the same throughout the period of the study (Figure 10F), and this provided further evidence that the ISF was extracted through the skin appendages but not the SC as it was unaffected by partial SC removal.

[0151] In the mouse melanoma model cytokine analysis revealed the increase of all 19 cytokines analysed in tumour lysate samples, 4 cytokines in serum, and 9 cytokines in ISF following tumour inoculation. Two approaches were used to compare the cytokines in the longitudinal samples. Firstly, any statistically different changes in the amount of cytokines at the time-points post tumour inoculation were compared to the pre-tumour inoculation levels. This involved normalising the ISF cytokine data per total micrograms (mcg) of protein in each ISF sample and thus the data was presented as picograms per mcg of protein (pg per mcg protein). This accounted for the different amounts of ISF that were extracted using the stretching device at each extraction point. Secondly, to allow summarisation of the results in a "heat map" fold changes were calculated. For ISF this involved dividing post-tumour ISF values by pre-tumour ISF values (healthy ISF). In the case of serum, levels were presented as pg / mL, and fold changes were calculated by dividing post-tumour serum levels by pre-tumour serum levels (healthy serum). One limitation of this study was the absence of a control skin sample for the tumour lysate; hence fold changes were calculated by dividing tumour lysate levels by pre-tumour ISF levels (healthy ISF).

[0152] Analysis of longitudinally collected ISF samples revealed a statistically significant increase (p > 0.05), at least one time-point, in the levels of IL-9, IL-6, MIP-1, MCP-1, IP-10, IL-10, IL-12p70, IL-15, and IL-33 post-tumour inoculation. IP-10, IL-33 and MCP-1 spiked on early stages (days 1-3). A spike in the remaining cytokines occurred generally 4 days after tumour inoculation. This coincided with the measurable and palpable tumours. On day 4, IL-33, IL-15, IL- 12p70, IL-6, IL-9, MIP-1, and MCP-1 reached levels of 0.018 ± 0.008, 0.58 ± 0.43, 0.16 ± 0.12, 0.04 ± 0.02, 0.26 ± 0.19, 0.031 ± 0.015, and 0.04 ± 0.02 pg per mcg protein, respectively (Figure 11).

[0153] When examining fold changes, it was observed that all fold changes for the two shared cytokines (IL-6 and MCP-1) were higher in the ISF compared to serum (Figure 12). For the remaining cytokines, all fold changes were higher in the tumour lysate than either serum or tumour lysate. Fold changes were presented as zero for any cytokine below LOD (Figure 12). These findings highlight ISF demonstrate a closer match to the tumour lysate's cytokine profile like the proteomics analysis.

[0154] More than 1500 proteins were identified from the ISF samples taken from the skin on the surface of the tumour using the Mouse Taxonomy database in the Scaffold proteomic software. After eliminating proteins that were already present in the ISF or serum of healthy mice and excluding proteins with fewer than 3 peptide spectral matches, 346 proteins were identified (Figure 13). The concept of peptide spectral matches refers to the total number of peptides identified in the mass spectrometry raw data that align with the peptides listed in the Scaffold database. It is generally accepted in scientific literature that a match is considered significant when at least three peptide spectral matches are identified.

[0155] The tumour lysate exhibited the highest number of proteins, with 297 proteins detected. The ISF shared more proteins, 45 proteins, with the tumour lysate compared to the serum, which shared 17 proteins with the tumour lysate (Figure 13). There were 15 common proteins between the ISF and serum samples (Figure 13). The absence of distinctive proteins in the serum can be attributed to the systemic circulation, which may dilute and homogenise unique proteins originating from the localised tumour microenvironment. To ensure accurate comparisons, the results were presented and analysed by normalising ion intensity values. Ion intensity values reflect the abundance of ions detected during sample analysis, but variations can occur between samples of different matrices, such as ISF and serum, necessitating the need for normalisation. The normalisation process involved adjusting the ion intensity values to allow for fair comparisons across samples, and it was achieved by normalising each sample to the lowest total protein content among the analysed samples. Results were displayed as normalised ion intensity values which were unitless.

[0156] A detailed examination of the 30 proteins shared between ISF and tumour lysate showed that most of the proteins exhibited an increase in abundance on the 7th day following tumour implantation (Figure 13). Notably, one of these proteins, lactate dehydrogenase (LDH), is recognised as a prognostic marker in the serum of melanoma patients. The analysis in the present study here demonstrated that LDH was absent in the serum of melanoma-bearing mice but exhibited a substantial increase on the 7th day in the ISF of these mice, reaching an intensity level of 3.9 x 108± 1.9 x 108before subsequently decreasing on the 10th day to an intensity level of 1.2 x 108± 7.5 x 107. Conversely, the S100 B protein displayed an opposing trend, with lower levels on the 7th day, recording a value of 4.8 x 107± 1.3 x 107, and then a notable spike on the 10th day, reaching 6.7 x 108± 9.3 x 108. The majority of other relevant melanoma proteins such as elongation factor, myosin, vimentin, and filaggrin spiked on day 7. Moreover, the two proteins that were in the ISF only are hornerin and odorant binding protein. Hornerin, an epidermal protein, is known to exhibit increased levels in inflammatory conditions, while odorant-binding protein, associated with skin and membrane inflammation, plays a role in olfaction. The unique presence of these two proteins in the ISF, particularly the epidermal protein hornerin, can support that the fluid extracted is ISF. Analysis of the 15 proteins shared among ISF, serum, and tumour lysate at the endpoint of the study (day 10, Figure 13) showed that the majority of the 15 proteins were found to be more abundant in tumour lysate compared to ISF and serum. Only two proteins exhibited similar levels across all three biofluids., which are histone and plastin 2. Three proteins were more abundant in ISF, progranulin, flavin reductase, and chloride channel protein. Therefore, the fluids draining the tumour tissue site had less proteins than in the tumour, they were in general less abundant, but ISF contained more proteins and in greater abundance than the serum.

[0157] Examole 5 - Effect of device size, extraction time and device orientation on extraction of skin ISF from humans

[0158] Methods

[0159] The studies were conducted under standardized conditions and participants were given time to adapt to room conditions prior to start of each study, which was conducted by a group of the same investigators. Prior to the ISF extraction, the skin sites were delineated with a marker pen and the skin surface contaminants were removed at each site by: 1) washing using soap and water, 2) wiping with 70% v / v ethanol and 3) the application and removal of two tape strips. Consent was obtained from all the study participants as per the HR / DP- 22 / 23-27131 and approved by the King's College London Research Ethics Committee. Pain scores were obtained from all participants for the application of skin stretching device. The study was conducted on the dorsal skin of the forearm of healthy male and female adult volunteers. A 150 pl aliquot of sterile PBS (pH 7.4) was added to the surface of the skin followed by skin stretching conditions obtained by the application of the 2-part 3D printed stretching device without a loading post at -4.5 psi for 10 min to extract ISF. After the 10 min extraction time, the extraction buffer was carefully collected, the volume determined, and the samples were stored at -80°C until analysis for total protein using the BCA assay as per the manufacturer's instructions.

[0160] Results

[0161] Using the 2-part device the larger device (orifice diameter of 26 mm) can extract more ISF compared to the medium sized device (orifice diameter of 20 mm) and the small size device (orifice diameter of 14 mm) (Figure 14). However, the larger the size of device the less efficient the extraction, shown by dividing the volume of ISF extracted by the surface area of the skin. Using the medium sized device, it was shown that an extraction time of 10 min was optimal and that repeated extraction can increase the ISF extracted (Figure 15). Using the one- part device ISF can be successfully extracted regardless of the arm orientation demonstrating the success of using a membrane / gauze to extract the ISF rather than a buffer (Figure 16).

[0162] Examole 6 - The use of the extraction device to monitor inflammation in humans

[0163] Methods

[0164] The study was conducted on healthy male and female adult volunteers (20-40 years old, n = 8). The extraction of the chemicals from the skin was conducted either without skin stretching ( atmospheric pressure conditions (0 PSI, 10 min) or using skin stretching obtained by the application of the two-part device without a loading post at -4.5 psi for 10 min. Low-grade inflammation was induced using the epidermal dysfunction model achieved by tape-stripping a 2 x 2 cm area of the forearm dorsal skin until 6 to 7 -fold increase in TWEL was achieved. Four sites of the dorsal forearm (2 sites on each arm) were used from each participant: 1) Arm 1, untreated skin with atmospheric or skin stretching extraction and 2) Arm 2, skin with ED with atmospheric or skin stretching extraction. TEWL at each site was measured prior to start of the study using a BIOX TEWL meter and then at 0, 3 h, 6 h and 24 h post the induction of ED. The ISF extraction was completed using sterile 3D printed chambers held on the skin using a medical double-sided adhesive (3M™ Medical Silicone Tape 2477P). The extraction was initiated by the application of a 150 pl aliquot of sterile PBS (pH 7.4) on the surface of the skin and the application of the hypobaric pressure. After the 10 min extraction time, the extraction buffer was carefully collected and stored at -80°C until analysis for pro-inflammatory cytokines using the S-Plex panel 1 (Meso Scale, US) and for protein markers using proteomics analysis (KCL, UK)

[0165] Skin acjincj

[0166] The study was conducted on the dorsal skin of the forearm of healthy male and female adult volunteers with different ages. The participants were divided into three age groups: Groupl - 20-40 y / o (n = 14), Group2- 40-60 y / o (n=ll) and Group3- 60-80 y / o (n = ll). A 150 pl aliquot of sterile PBS (pH 7.4) was added to the surface of the skin followed by skin stretching conditions obtained by the application of -4.5 psi for 10 min to extract ISF. After the 10 min extraction time, the extraction buffer was carefully collected, the volume determined, and the samples were stored at -80°C until analysis for total protein with the BCA assay, pro-inflammatory cytokines using the S-Plex panel 1 (Meso Scale, US) and for protein markers using proteomics analysis (KCL, UK).

[0167] In all the human volunteers, the TEWL was measured using a AquaFlux Model AF200 TEWL meter (Biox Systems Ltd, UK) at the dorsal forearm under untreated conditions (baseline), then after the application of skin stretching. For ED study, TEWL was measured prior the induction of the ED, then at predetermined time points up to 24 h.

[0168] Skin Elasticity measurement

[0169] Skin elasticity in all the human volunteers was measured using a Cutometer®MPA580 (Courage + Khazaka, Cologne, Germany) on the dorsal side of the forearm under untreated conditions. The skin elasticity measurements were performed with a 6-mm-diameter probe was used at a pressure of -450 mbar with 5 s of suction followed by 5 s of release, repeated three times. This was performed on three different sites.

[0170] Total D rotein assessment

[0171] The total protein concentration of extracted samples was assessed used the Pierce BCA Protein Assay (ThermoFisher, UK) according to manufacturer's instructions. Briefly, the working solution was made by through mixing of stock solutions A and B at a ratio of 50: 1. The test sample (10 pl) was added the well of a 96 well plate, followed by 200 pl of the working stock solution. The plate was placed on a shaker for 1 minute at 500 RMP to allow thorough mixing. The plate was the incubated at 37 °C for 30 minutes using a pre-heated oven, before leaving to cool at room temperature for 20 minutes. The absorbance of each well was then measured at 562nm using a Tecan Spark plate reader (Tecan Ltd, UK).

[0172] The study employed a S-Plex Proinflammatory Panel 1 Multiplex Kit (Mesoscale Diagnostics, Maryland, USA) to measure cytokines in the extracted ISF. This kit facilitated the simultaneous analysis of various cytokines, including IFN-y, IL-ip, IL-2, IL-4, IL-6, IL-17, IL-10, IL-12p70, and TNFo. The assay was conducted in a 96-well plate, coated prior to use and incubated at room temperature with shaking. Calibration standards were prepared using a multianalyte lyophilized calibrator, following a 4-fold serial dilution. After incubation, wells were washed, blocked, and loaded with 25 pl samples or standards. Subsequent steps included additional washes, the addition of antibody and enhance solutions, and a final detection solution step. The plate was then analysed in the MSD Meso Quickplex SQ 120 instrument (Mesoscale DiagnosticsTM, Maryland, USA). MSD Mesoscale Discovery Workbench™ 4.0 software was employed for data analysis, providing cytokine concentrations in femtograms per milliliter (fg / mL) for each well.

[0173] Results

[0174] The removal of the stratum corneum to induce ED theoretically leads to the release of TNF-alpha and this then induces the release of local cytokines in the skin (Figure 17A). Applying the device to the skin did not cause any visible damage to the tissue either with the healthy skin or the ED skin (Figure 17B). Of the cytokines that recorded measurable levels in the skin extraction fluid (IL1- beta, IL6, TNF-alpha) only IL6 showed a measurable increase during the 24 h experiment (Figure 17 C,D,E). The proteomics analysis showed changes in 17 proteins compared to the control tissues (Figure 17 F, G).

[0175] In the inflammation study that investigated chronic intrinsic inflammatory cytokine release, the youngest age group aged 20-40 years old showed lower levels of IL- ip and IL-17 when compared to the oldest age group 60-80 years old (Figure 18). The levels of IL-ip and IL-17 were above LOD and LOQ for the multiplex MSD analysis. However, all remaining cytokines were below LOD. The youngest age group had IL- ip levels of 2.44 ± 2.45 pg / pL of ISF extracted while the oldest age group had IL-ip levels of 9.85 ± 9.74 pg / pL of ISF extracted. Moreover, the youngest age group had IL-17 levels of 0.0006 ± 0.002 pg / pL of ISF extracted while the oldest age group had IL-17 levels of 0.008 ± 0.01 pg / pL of ISF extracted. The TEWL levels were the same across all age groups, the youngest age group recorded 19.29 ± 5.60 g / m2h, while the middle age group had TEWL levels of 23.99 ± 6.68 g / m2h, and the oldest age group had levels of 21.57 ± 6.25 g / m2h. to modify the skin

[0176] Methods

[0177] The ISF extraction studies were conducted under standardized conditions and participants were given time to adapt to room conditions prior to start of each study which was conducted by the same investigators. Prior to the ISF extraction, the skin sites were delineated and the skin surface contaminants were removed at each site by: 1) washing using soap and water, 2) wiping with 70% v / v ethanol and 3) the application and removal of two tape strips. Consent was obtained from all the study participants as per the HR / DP-22 / 23-27131 and approved by the King's College London Research Ethics Committee. Pain scores were obtained from all participants for the application of skin stretching device. The study was conducted on the dorsal skin of the forearm of healthy male and female adult volunteers. A 150 pl aliquot of sterile PBS (pH 7.4) was added to the surface of the skin followed by skin stretching conditions obtained by the application of a 3D printed 2-part device with and without a loading -4.5 psi for 10 min to extract ISF. After the 10 min extraction time, the extraction buffer was carefully collected, the volume determined, and the samples were stored at -80°C until analysis for total protein using the BCA assay as per the manufacturer's instructions.

[0178] Results

[0179] The results showed if a "post" was used in the centre of the device that enabled the dome formation to be supressed and the skin stretched laterally, up to 4 pl of ISF can be sampled from the skin (Figure 19). The surprising capacity of the device to extract a large volume of ISF without damage to the skin allows repeated sampling from the same skin site, which is needed in many medical applications and is unique to the device.

[0180] When the dimensions and the type of the loading post we modified it was found that a long loading post which almost touched the skin with 17 mm diameter (device orifice diameter was 20 mm) was optimal to extract the ISF. Using concentric circles to minimise the skin pinching effect around the outer sides of the device was also effective (Figures, 19 and 20).

[0181] Examole 8 - The imoact of usina a loadina Dost on biomarker extraction from the skin Methods

[0182] Human ISF extraction using skin stretching

[0183] Extraction was performed with either the standard 3D printed 2-part device or the one with the 17 mm loading post in for 10 minutes at -4.5 psi using a collection buffer volume of 150 pl. Samples were then assessed for protein concentration with the BCA assay and the identification of small molecular weight biomarkers was performed using gas chromatography mass spectrometry or cytokines were analysed using MSD ultrasensitive S-plex proinflammatory cytokine multiplex plate.

[0184] GC-MS analysis

[0185] Human ISF samples were collected by using 2 different 3D printed devices, which were the devices with and without loading post. Only buffer from the skin was also collected as a control under atmospheric conditions. Blood serum was collected from participants. (Blood data will be added later). The samples were stored under -80 until analysis. Then 150 pl cooled extraction solvent was added to one 1.5 mL microfuge tube containing 150 pl of the freshly thawed ISF. This was vortexed for 10 sec and on Thermomixer shake for 30 min at 4°C (1000 rpm shaking needed) and then centrifuged for 30 min at 14,000 rpm, room temperature. The samples were evaporated and resuspended in the 150 pL nitrogen-degassed 1 : 1 (v / v) acetonitrile / IPL at room temperature. Next, the samples were centrifuged for 30 min at 14,000 rpm, room temperature. Supernatants were taken into a new 1.5-ml polypropylene microcentrifuge tube. Then for each sample, 6 pl of 25-Hydroxyvitamin D3 was added and then dried with a gentle stream of nitrogen. (Amount of internal standard: 0.3 pg). This will be followed by adding 1 pL of MeOX solution to an autosampler vial and mixing for 90 min at 30C. Then, 9 pL of MSTFA (N-Trimethylsilyl-N-methyl trifluoroacetamide) +FLORIDA mixture was added to the autosampler vial. (97.5 pl of MSTFA+2.5 pl of FLORIDA. Vortex the solution). The final concentration of FLORIDA: 10 pg / ml. (Stock FLORIDA: 500 pg / ml). The final concentration of Deuterated Vitamin D: 30 pg / ml (Stock deut Vitamin D: 50 pg / ml). The mixture is completely mixed and incubated samples at 60°C for 60 minutes. Submit to GC- MS.

[0186] After RT and derivatization check for each injection, samples were analyzed. These steps include (1) spectrum deconvolution, to convert raw data into a peak list; (2) metabolite identification, to recognize metabolites associated with chromatographic peaks; (3) quantification, to compare the abundance of a specific metabolite in different samples; (4) association network analysis, to reveal correlations among the changes in the abundance of multiple metabolites; and (5) pathway analysis, to understand the biochemical interrelationship between several metabolites that vary in a coordinated or differential manner.

[0187] The study employed a S-Plex Proinflammatory Panel 1 Multiplex Kit (Mesoscale Diagnostics, Maryland, USA) to measure cytokines in the extracted ISF. This kit facilitated the simultaneous analysis of various cytokines, including IFN-y, IL-ip, IL-2, IL-4, IL-6, IL-17, IL-10, IL-12p70, and TNFo. The assay was conducted in a 96-well plate, coated prior to use and incubated at room temperature with shaking. Calibration standards were prepared using a multianalyte lyophilized calibrator, following a 4-fold serial dilution. After incubation, wells were washed, blocked, and loaded with 25 pl samples or standards. Subsequent steps included additional washes, the addition of antibody and enhance solutions, and a final detection solution step. The plate was then analysed in the MSD Meso Quickplex SQ 120 instrument (Mesoscale DiagnosticsTM, Maryland, USA). MSD Mesoscale Discovery Workbench™ 4.0 software was employed for data analysis, providing cytokine concentrations in femtograms per milliliter (fg / mL) for each well.

[0188] Results

[0189] The most important small molecular weight compounds identified in the ISF using GC-MS were compared and listed in Figure 21. Most of the compounds detected using the ISF from the standard 2-part device (HYP) were not detected in the control group (ATM). There were more compounds detected in the ISF when the two-part device using a loading post was employed (HYP - loading post). This suggests that the 2-part loading post device is more successful at extracting small molecular weight chemicals from the skin.

[0190] When using the two part device with the loading post to extract ISF from the skin there was a statistically significant increase in all but one of the cytokines tested compared to the 2-part device without the loading post. Most cytokines were not detected with the standard 2-part device; however, all were detected in the loading post samples. There was also a significant increase in the recovered amount of ISF, with around 2-3x more ISF in loading post samples. This suggests the loading post design is more successful at extracting large molecules from the skin.

[0191] Examole 9 - The imoact of the device material on tolerabilitv of the extraction device

[0192] Methods

[0193] Utilizing 3D printing technology, a range of biocompatible resins were employed to determine the most suitable material for the ISF device. The test materials included Flexible 80A, a resin known for its durability and flexibility; BioMed Clear, which offers a balance of strength and clarity. These commercially available materials were adapted by adding different percentages of PEG 400 to generate BioMed Clear 80 and BioMed Clear 50, which were more flexible and allowed for softer contact with the skin, (Figure 23).

[0194] To evaluate the tolerability of the materials used in the one-part extraction device a series of tests were conducted that measured participant discomfort levels using a pain scoring sheet. Participants were subjected to device application for intervals of 5 or 10 minutes under varying degrees of negative pressure. Upon completion of each test, participants rated the discomfort experienced through the pain scores. The results from these pain scores were then analysed by plotting the pain vs the suction pressure and measuring the area under the curve to ascertain the most suitable material and pressure settings that would allow the device to function effectively while maintaining patient comfort.

[0195] Results

[0196] In the assessment of device material tolerability for a one-part extraction device, the area under the curve (AUC) was inversely proportional to patient comfort, with lower AUC values indicating reduced discomfort. Flexible 80A demonstrated the most favourable tolerability profile, exhibiting the lowest AUC values at both 5 and 10 min application times, indicative of minimal patient- reported pain scores, (Figure 23).

[0197] At the 5-minute interval, Flexible 80A showed significantly superior tolerability compared to all concentrations of BioMed Clear, with a lower AUC indicating reduced discomfort (p < 0.05). This trend persisted at the 10-minute application, where Flexible 80A maintained its position as the most comfortable material, as evidenced by the AUC remaining the lowest among the materials tested, although the differences were not statistically significant at this interval (p > 0.05).

[0198] BioMed Clear at 65% and 80% concentrations showed an intermediate level of comfort, with their AUC values being higher than Flexible 80A but not significantly different from each other at both time points. In contrast, BioMed Clear at 100% concentration reported the highest discomfort levels at 5 minutes, which did not increase significantly at 10 minutes, suggesting a plateau in discomfort perception over time. These results underscore Flexible 80A as the most comfortable material for the one-part extraction device, demonstrating a consistent tolerability advantage over the range of BioMed Clear concentrations across the application times tested.

[0199] Example 10 - Using the loading Dost device to extract ISF from animals

[0200] Methods

[0201] To take ISF samples from the mice they were anaesthetised by inhalation of (1-2%) isoflurane I (1-2 %) O2 and placed on a heating mat (Harvard Apparatus, Cambridge, UK) in the ventral position maintained at 37 °C throughout the experiments. The dorsal hair was carefully removed (to avoid any damage to the skin) with an animal hair clipper (Centura, London, UK). The 3D printed device was placed on the lower flank of the mice, and 150 pL sterile PBS (pH 7.4) was introduced into the device via the top of the tubular structure, making direct contact with the mouse skin. Different pressures were applied for between 2-20 min via an in-house developed automated pump that self-corrected in case of pressure loss. At the end of the experiment the pressure was released, and PBS in contact with the skin was carefully collected using a pipette. The skin area was monitored for any persistent redness within 24 h. The amount of ISF extracted was determined by measuring the total protein in the sample using the BCA assay kit as per the manufacturer's instructions.

[0202] Results

[0203] Three different devices were constructed. The three-part devices were preferred as they stabilised the mouse skin during the application of the pressure (Figure 24).

[0204] The 3-part device proved to be only tolerated up to 15 min at -4.5 psi and for only 10 min at 5.5 psi using the loading post (Figure 25). When the ability of the 3-part device to extract ISF from the mice was compared it showed that the addition of the loading post increased the ISF extraction by up to 3-fold (Figure 26).

[0205] Examole 11 - Continuous extraction of ISF usina the loadina Dost device

[0206] Methods

[0207] The ISF extraction studies were conducted under standardized conditions and participants were given time to adapt to room conditions prior to start of each study which was conducted by the same investigators. Prior to the ISF extraction, the skin sites were delineated and the skin surface contaminants were removed at each site by: 1) washing using soap and water, 2) wiping with 70% v / v ethanol and 3) the application and removal of two tape strips. Pain scores were obtained from all participants for the application of skin stretching device. The study was conducted on the dorsal skin of the forearm of healthy male and female adult volunteers. A 150 pl aliquot of sterile PBS (pH 7.4) was added to the surface of the skin followed by skin stretching conditions obtained by the application of a 3D printed 2-part device with and without a loading post at -4.5 psi for different time periods to extract ISF. After the 10 min extraction time, the extraction buffer was carefully collected, the volume determined, and the samples were stored at -80°C until analysis for total protein using the BCA assay as per the manufacturer's instructions. The total protein was converted into amount of ISF given a standardised amount of protein in the body of a human adult.

[0208] Results

[0209] The device without a loading post allowed a maximum of 1 pl of ISF to be extracted, with no benefit above 2.5 minutes, showing this device does not allow continuous extraction. More ISF was extracted at all time points using the loading post device (Figure 27).

[0210] Examole 12 - Use of a honevcomb collection aauze with the loadina Dost device

[0211] Methods

[0212] The ISF extraction was completed as per Example 11 but at one extraction time, 10 min, at one pressure -4.5 psi. In addition, the loading post device was modified by the addition of a honeycomb collection mesh that was designed and printed using an Asiga 3D printer with clear resin. Dimensions of the mesh and process for collection are outlined in Figure 28. A device was made to allow the mesh to be centrifuged to separate liquid from the mesh for downstream analysis (Figure 28). Recovery volume was determined immediately after loading and after extraction. Extraction samples were used for protein quantification by BCA assay.

[0213] Results

[0214] ISF extraction samples with the mesh from humans were quantified for protein content with the BCA assay and compared to the surgical gauze collection and the "standard" 2-part loading post device where the ISF is collected in a collection buffer. ISF volume was calculated based on the theoretical maximum volume held by the mesh of 80.9 pl. This showed an average recovery of 1.34 pl of ISF demonstrating that such an approach was promising to collect the ISF without the need for a dilution buffer (Figure 27). This approach could also be useful for concentrating the ISF liquid on the surface of a sensor that could be inserted into the device.

[0215] 13 - Delivery into the skin using the device

[0216] Methods

[0217] FITC-dextran purification prior to permeation studies

[0218] FITC-dextran 150 kDa was purified using the twelve consecutive cycles of ultra-filtration method (described above) using ultra-filtration (Amicon filters, 100 kDa MWCO). The obtained final solution was then analysed using gel filtration size exclusion chromatography to confirm the absence of any free FITC label.

[0219] Skin permeation and deposition

[0220] Porcine ear skin samples were obtained from local butcher (The Ginger Pig, Borough Market, London) and were stored and wrapped in aluminium foil at -20 °C until use. Full thickness skin was prepared by the physical removal of the hypodermis using a scalpel. The prepared skin was washed with de-ionised and blotted dry before use.

[0221] Permeation of rhodamine dye and the purified macromolecules (FITC- dextran 150 kDa) across full thickness porcine ear skin was conducted using upright individually calibrated Franz diffusion cells for 1 h at 32 °C (water bath maintained at 37 °C) using hypobaric pressure conditions (-4.5 psi). The Franz cells were assembled as described previously (5). Hypobaric pressure was produced using in-house 3D-printed pressure chambers attached to a hand pump (allowing the adjustment of pressure) (equivalent to the 1 part human device). The study was initiated by the application of an infinite dose (150 uL - 1000 uL) of the rhodamine or purified FITC-dextran (100 pg / ml) in PBS (pH 7.4) solutions and subsequent induction of hypobaric pressure conditions. The hypobaric pressure was maintained at -4.5 psi for the duration of 1 h. For this period, a wet sponge disc (PBS, pH 7.4) was placed underneath the skin and acted as the receiver compartment (in order to prevent extraction of receiver fluid into the donor chamber). At the end of the 1 h hypobaric pressure application, the sponge disc was removed, and the rhodamine or FITC-dextran was extracted and quantified.

[0222] Analytical assay

[0223] Quantitative determination of rhodamine and FITC-dextran in the skin permeation and deposition studies and for the purification studies was performed using a Tecan Spark plate reader (Tecan Ltd, UK) at excitation wavelength of 485 nm and emission wavelength of 535 ± 20 nm. The diluent used was identical to that used for the permeation and purification studies (i.e. PBS, pH 7.4) and the volume of the solution per well was 200 pl. The calibration curves were constructed according to fluorescence intensity measurements.

[0224] Results

[0225] Use of a sponge or gauze with the loading post device, or the use of a loading post device with a reservoir (with or without a sponge) allowed the test compound to stay in contact with skin better than the standard loading post device (Figures 30 and 31).

[0226] References

[0227] 1. Ahn NG, Shabb JB, Old WM, Resing KA. Achieving in-depth proteomics profiling by mass spectrometry. ACS Chem Biol. 2007 Jan 23;2(l):39-52.

[0228] 2. Samant PP, Niedzwiecki MM, Raviele N, Tran V, Mena-Lapaix J, Walker DI, Felner El, Jones DP, Miller GW, Prausnitz MR. Sampling interstitial fluid from human skin using a microneedle patch. Sci Transl Med. 2020 Nov 25;12(571):eaaw0285.

[0229] 3. Takematsu H, Tagami H. Determination of released CD4 and CD8 antigens in the suction-blister fluid and horny-tissue extract in patients with psoriasis. Br J Dermatol. 1991 Jun;124(6):550-4. 4. Zuber TJ. Punch biopsy of the skin. Am Fam Physician. 2002 Mar 15;65(6): 1155-8, 1161-2, 1164.

[0230] 5. Inacio R, Poland S, Cai XJ, Cleary SJ, Ameer-Beg S, Keeble J, Jones SA. The application of local hypobaric pressure - A novel means to enhance macromolecule entry into the skin. J Control Release. 2016 Mar 28;226:66-76.

[0231] 6. Benaouda, F., Inacio, R., Lim, C. H., Park, H., Pitcher, T., Alhnan, M. A., ... & Jones, S. A. (2022). Needleless administration of advanced therapies into the skin via the appendages using a hypobaric patch. Proceedings of the National Academy of Sciences, 119(18).

[0232] 7. Sopasakis, V.R.; Smith, N.U. Cytokine release from adipose tissue of nonobese individuals. Int J Obes (Lond). 2005.29(9) : 1144-7.

[0233] 8. Thalmann, S.; Juge-Aubry, C.E.; Meier, C.A. Explant cultures of white adipose tissue. Methods Mol Biol. 2008. 456: 195-9.

[0234] 9. Eng JK, McCormack AL, Yates JR. An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database. J Am Soc Mass Spectrom. 1994 Nov;5(ll):976-89.

[0235] 10. Hu, LZ.; Mauro, TM.; Dang, EL.; Man, G.; Zhang, J.; Lee, DL.; Wang, G.; Feingold, KR.; Elias, PM.; Man, MQ. Epidermal Dysfunction Leads to an Age- Associated Increase in Levels of Serum Inflammatory Cytokines. Journal of Investigative Dermatology. 2017. 137(6): 1277-1285.

Claims

Claims1. A skin permeabilisation device comprising: a chamber to be placed in contact with a skin tissue surface of a subject and capable of forming a hypobaric seal with the skin tissue surface to provide a permeabilisation surface sealed within the chamber, at least one protrusion positioned within the chamber and arranged to contact a majority of the permeabilisation surface to restrict doming of the permeabilisation surface.

2. The skin permeabilisation device according to claim 1, wherein the at least one protrusion is arranged to contact at least about 75% of the permeabilisation surface.

3. The skin permeabilisation device according to claim 2, wherein the at least one protrusion is arranged to contact at least about 90% of the permeabilisation surface.

4. The skin permeabilisation device according to any preceding claim, wherein the at least one protrusion and the chamber are coaxial.

5. The skin permeabilisation device according to any preceding claim, wherein the at least one protrusion comprises a contact face that is arranged to contact the permeabilisation surface to restrict doming of the permeabilisation surface.

6. The skin permeabilisation device according to claim 5, wherein the contact face has a longest dimension of from about 5 mm to about 100 mm.

7. The skin permeabilisation device according to claim 5 or claim 6, wherein the contact face comprises a relief pattern of concentric circles.

8. The skin permeabilisation device according to any preceding claim, wherein the at least one protrusion is substantially cylindrical.

9. The skin permeabilisation device according to any preceding claim, wherein the subject is a mammalian subject.

10. The skin permeabilisation device according to any preceding claim, wherein the subject is a human subject.

11. The skin permeabilisation device according to any preceding claim, wherein the chamber is arranged to receive extraction fluid.

12. The skin permeabilisation device according to any preceding claim, wherein the skin permeabilisation device is of unitary construction.

13. The skin permeabilisation device according to any one of claims 1-11, wherein the at least one protrusion is removable from the chamber.

14. The skin permeabilisation device according to any preceding claim, wherein the skin permeabilisation device is for extraction of skin interstitial fluid from the skin tissue.

15. The skin permeabilisation device according to any preceding claim, wherein the skin permeabilisation device is for delivery of an agent to the skin tissue.

16. The skin permeabilisation device according to claim 15, wherein the agent is a biomolecule.

17. A method of obtaining interstitial fluid from a skin tissue surface of a subject, wherein the method comprises: applying the skin permeabilisation device according to any preceding claim to a skin tissue surface of a subject; applying negative pressure to the chamber such that the chamber forms a hypobaric seal with the skin tissue surface and interstitial fluid is extracted from the permeabilisation surface into the chamber, wherein the at least one protrusion contacts a majority of the permeabilisation surface to restrict doming of the permeabilisation surface; and collecting interstitial fluid from the chamber.

18. The method according to claim 17, wherein -3.5 psi to -15 psi pressure is applied to the chamber.

19. The method according to claim 17 or claim 18, wherein negative pressure is applied for 5 minutes to 15 minutes.

20. The method according to any one of claims 17-19, wherein the subject is a human subject.

21. The method according to any one of claims 17-20, further comprising depositing an absorbent substrate within the chamber prior to interstitial fluid extraction, and collecting the absorbent substrate from the chamber after interstitial fluid extraction.

22. A method of delivering an agent to a skin tissue of a subject, wherein the method comprises: applying the skin permeabilisation device according to any preceding claim to a skin tissue surface of a subject; introducing an agent for delivery to the skin tissue into the chamber; applying negative pressure to the chamber such that the chamber forms a hypobaric seal with the skin tissue surface and the agent is delivered through the permeabilisation surface into the skin tissue, wherein the at least one protrusion contacts a majority of the permeabilisation surface to restrict doming of the permeabilisation surface.

23. The method according to claim 22, wherein the agent is a biomolecule.

24. The method according to claim 23, wherein the biomolecule has a molecular weight of 10 kDa or more.

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