Methods and systems for enhanced body fluid collection

Abstract

Methods and systems are provided for collecting interstitial fluid (ISF) from a subject. The method includes applying energy' into a selected region of skin effective to facilitate ISF mobilization to and / or within the selected region; inserting an array of microneedles into the skin at an insertion site to form a plurality of micropores in the skin in the selected region; and collecting ISF from the insertion site. The systems includes an energy source configured to deposit energy7 into a selected region of the skin effective to facilitate ISF mobilization within the region; an array of microneedles configured for insertion into the skin to form a plurality of micropores therein in the selected region; a device configured to induce a pressure gradient to further mobilize ISF and to withdraw ISF through the micropores; and means for collecting the ISF that has flowed from the micropores to the surface of the skin.

Description

17625-0344 GTRC 2024-159 METHODS AND SYSTEMS FOR ENHANCED BODY FLUID COLLECTION Cross-Reference to Related Applications

[0001] This application claims priority to and benefit of U.S. Provisional Patent Application No.63 / 683,893, filed August 16, 2024, which is incorporated by reference herein in its entirety. Statement Regarding Federally Sponsored Research or Development

[0002] This invention was made with government support under Contract Numbers FA8650-18-2-5402 and FA8650-15-2-5401 awarded by the Air Force Research Laboratory. The government has certain rights in the invention. Background

[0003] Interstitial fluid (ISF) in skin is rich in biomarkers with potential use in diagnostics and monitoring for both dermal and systemic indications. ISF offers advantages over blood in that it does not clot, which facilitates continuous or repeated monitoring, has no cells and low albumin content, which simplifies sample preparation for analysis, and contains unique and unexplored biomarkers not found in blood that can provide tissue-specific information.

[0004] The only biomarker routinely monitored in ISF is glucose by diabetic patients, which is measured by a sensor implanted in the subcutaneous space. To avoid an indwelling sensor, various methods have been used to collect biomarkers from dermal ISF for analysis outside skin, including microdialysis, open-flow microperfusion, laser photoporation, reverse iontophoresis, low-frequency ultrasound, and microneedles, but these methods suffer from being complex, invasive, costly and / or able to collect only small amounts of ISF biomarkers.

[0005] Microneedle-based methods have received increasing attention because of their simplicity and low cost. Microneedles are typically solid, hollow, or hydrogel structures measuring hundreds of microns long that pierce skin to access dermal ISF. Fluid can be collected from skin by diffusion, capillary action, pressure / suction or other driving forces. Metabolomic and proteomic analyses of ISF extracted using microneedles have established that most compounds found in blood are also present in ISF, and that ISF also contains thousands of compounds not found in blood. However, widespread use of ISF extracted using microneedles in clinical research and medicine has been limited by the small ISF volumes they collect, usually just up to a few microliters.

[0006] Thus, there remains a need for similarly minimally-invasive methods of collecting ISF from skin that are capable of collecting large volumes of ISF. 1 53430309.117625-0344 GTRC 2024-159 Brief Summary

[0007] In one aspect, a method is provided that includes applying energy into a selected region of a subject’s skin effective to facilitate interstitial fluid (ISF) mobilization to, and / or within, the selected region; inserting an array of microneedles into the subject’s skin at an insertion site to form a plurality of micropores in the subject’s skin in the selected region; and collecting ISF from the insertion site. The subject may be a human, for example a human in need of diagnosis or monitoring.

[0008] In another aspect, a system is provided for collecting interstitial fluid (ISF) from a subject's skin, wherein the system includes: an energy source configured to deposit energy into a selected region of a subject's skin effective to facilitate ISF mobilization within the region; an array of microneedles configured to be inserted into the subject’s skin to form a plurality of micropores in the subject's skin in the selected region; a device configured to induce a pressure gradient, via positive pressure or suction, to further mobilize ISF and to withdraw ISF through the plurality of micropores formed by the array of microneedles; and means for collecting the ISF that has flowed from the micropores to the surface of the subject’s skin.

[0009] In still another aspect, a method is provide for detection and / or monitoring of disease, injury, environmental exposure, or physiological or drug pharmacokinetic status in a subject, the method including: collecting interstitial fluid (ISF) from the skin of the subject in accordance with the methods and / or systems described herein; and analyzing the collected ISF for the presence or concentration of (i) a biomarker indicative of a specific disease injury, environmental exposure, or physiological status, (ii) a pharmaceutical compound, (iii) a metabolite, or (iv) a combination thereof. Brief Description of the Drawings

[0010] The detailed description is set forth with reference to the accompanying drawings. The use of the same reference numerals may indicate similar or identical terms. Various embodiments may utilize elements and / or components other than those illustrated in the drawings, and some elements and / or components may not be present in various embodiments. Elements and / or components are not necessarily drawn to scale.

[0011] FIG.1A is a schematic representation of pressure application via suction, where suction is applied uniformly across an area of microneedle skin puncture.

[0012] FIG.1B is a schematic representation of pressure application via positive pressure, where positive pressure is applied in a ring around an area of microneedle skin puncture. 2 53430309.117625-0344 GTRC 2024-159

[0013] FIG.2 is a schematic representation of the physical basis for the model used to predict hydraulic conductivity of dermis as a function of water content, showing the macroscale, mesoscale, and microscale.

[0014] FIG.3 depicts a sampling chamber of an apparatus for measuring the hydraulic conductivity of the dermis and the skin.

[0015] FIG.4 depicts an apparatus for measuring the hydraulic conductivity of the dermis and the skin ex vivo.

[0016] FIG.5 is a graph of the modeled volume of ISF crossing the dermis based on modeled hydraulic conductivity for a skin sample at 70% hydration.

[0017] FIGS.6A-6F are graphs depicting the effect of pressure on hydraulic conductivity for individual ex vivo pig dermis samples sampled at 30-minute intervals.

[0018] FIG.7A depicts a microneedle array with 750 µm long microneedles compared to a standard 21G hypodermic needle.

[0019] FIG.7B is a magnified view of (i) a single 750 µm long microneedle, and (ii) a single 250 µm long microneedle.

[0020] FIG.7C is a graph depicting ISF extraction from ex vivo pig skin as a function of suction pressure.

[0021] FIG.7D is a graph depicting ISF extraction from ex vivo pig skin as a function of positive pressure.

[0022] FIG.8A is a graph depicting the effect of microneedle pretreatment on ISF extracted during application of 800 kPa positive pressure to ex vivo pig skin.

[0023] FIG.8B is a graph depicting the effect of microneedle length on ISF extracted during application of -50 kPa suction after pretreating ex vivo pig skin with microneedles.

[0024] FIG.8C is a graph depicting the effect of the type of suction pump on ISF extracted during application of -65 kPa suction to ex vivo pig skin.

[0025] FIG.8D is a graph depicting the effect of skin stretching on ISF extracted over a range of positive pressures applied to ex vivo pig skin.

[0026] FIG.8E is a graph depicting the effect of skin incubation temperature on ISF extracted at two different suction pressures applied to ex vivo pig skin.

[0027] FIG.8F is a plot depicting the effect of skin thickness on ISF extracted from ex vivo pig skin samples of various skin thickness.

[0028] FIG.9A is a graph depicting the water content of dehydrated ex vivo pig skin at different times, showing the original water content as compared to the water content after dehydrating. 3 53430309.117625-0344 GTRC 2024-159

[0029] FIG.9B is a graph depicting ISF extracted from the dehydrated ex vivo pig skin samples of FIG.9A, normalized to ISF volume extracted from a non-dehydrated skin sample.

[0030] FIG.9C is a plot of volume of ISF extracted from the dehydrated ex vivo pig skin samples of FIGS.9A-9B, plotted as a function of skin water content.

[0031] FIG.10A is a graph depicting the effect of enzyme concentration during skin incubation on ISF extracted from ex vivo pig skin treated with one of three different enzymes: collagenase type 1, hyaluronidase from bovine testes, and hyaluronidase from sheep testes.

[0032] FIG.10B is a graph depicting the effect of combined enzyme treatment on ISF extracted from ex vivo pig skin treated with hyaluronidase, collagenase, or a combination of hyaluronidase and collagenase.

[0033] FIG.10C is a graph depicting the effect of heating collagenase on ISF extracted from ex vivo pig skin incubated with or without collagenase.

[0034] FIG.10D is a graph depicting the effect of collagenase type on ISF extracted from ex vivo pig skin treated by incubating in different collagenase formulations.

[0035] FIG.11 is a graph depicting the sensitivity analysis of parameters in the fiber-matrix model on predicted hydraulic conductivity of skin, comparing changes in skin hydraulic conductivity after increasing or decreasing model parameters by 50% relative to base values.

[0036] FIG.12A is a graph comparing the effective hydraulic conductivity at 200 kPa transdermal pressure for full-thickness ex vivo pig skin compared to effective hydraulic conductivity of ex vivo pig dermis at the same pressure.

[0037] FIG.12B is a graph comparing water content of ex vivo pig dermis samples before and after dehydration.

[0038] FIG.12C is a plot of the effect of pressure on effective hydraulic conductivity of ex vivo pig dermis.

[0039] FIG.12D is a graph comparing the effect of water content on volume of fluid flow through ex vivo pig dermis (i.e., effective hydraulic conductivity) before and after dehydration.

[0040] FIG.13A is a graph comparing the mass of ex vivo pig dermis samples before and after dehydration.

[0041] FIG.13B is a graph comparing the effective hydraulic conductivity of ex vivo pig dermis samples when hydrated and dehydrated.

[0042] FIG.14 is a graph plotting the relationship between absolute melting temperature differential and pore size for the melting of water confined in small pores, plotted on a log scale.

[0043] FIG.15A depicts differential scanning calorimetry (DSC) thermograms of a single ex vivo pig skin sample with different levels of water content. 4 53430309.117625-0344 GTRC 2024-159

[0044] FIG.15B depicts cumulative enthalpy changes for the thermograms of FIG.15A.

[0045] FIG.15C is a graph showing the effect of skin water content on percent freezable and non-freezable water content and enthalpy changes of ex vivo pig skin samples.

[0046] FIG.15D is a graph showing the effect of skin water content on pore size distribution of ex vivo pig skin samples calculated based on the melting point of water in DSC thermograms.

[0047] FIG.15E is a graph showing the effect of ex vivo pig skin water content on median pore size of skin.

[0048] FIG.16A depicts thermograms from three different ex vivo pig skin samples scanned from -60 ºC to 5 ºC.

[0049] FIG.16B depicts thermograms for one of the skin samples of FIG.16A, subjected to four freeze-thaw cycles in the DSC.

[0050] FIG.16C depicts the cumulative enthalpy for the thermograms of FIG.16B.

[0051] FIG.16D is a graph showing the pore size distribution of freezable water in the skin after up to four freeze-thaw cycles for the samples of FIG.16A.

[0052] FIG.17A is a graphical representation of the number of laser micropulses measured at low laser energy settings.

[0053] FIG.17B is a graphical representation of the number of laser micropulses measured at medium laser energy settings.

[0054] FIG.17C is a graphical representation of the number of laser micropulses measured at high laser energy settings.

[0055] FIG.18A is a graph of the average energy emitted per laser micropulse at low, medium, and high laser energy settings.

[0056] FIG.18B is a graph of the number of pulses emitted by the laser at low, medium, and high laser energy settings.

[0057] FIG.19 is an optical coherence tomography image of the skin of a human subject after application of eight laser pulses.

[0058] FIG.20 is an optical coherence tomography image of the skin of a human subject immediately after application of a single laser pulse.

[0059] FIGS.21A-21C depict the increased water content produced by application of eight laser pulses to in vivo rat skin.

[0060] FIG.22A is a graph depicting the effect of laser treatment on ISF extracted from ex vivo pig skin. 5 53430309.117625-0344 GTRC 2024-159

[0061] FIG.22B is a graph depicting the effect of the number of laser pulses on ISF extracted from the skin of human subjects, each receiving laser treatment, microneedle treatment to generate 100 micropores, and suction for 5 minutes to extract ISF.

[0062] FIG.22C is a graph depicting pain scores for the treatments applied in FIG.22B.

[0063] FIG.22D is a graph depicting the effect of the duration of suction on ISF extracted from the skin of human subjects, each receiving laser treatment, microneedle treatment to generate 200 micropores, and suction for two periods of 5 minutes and one period of 10 minutes, or a single 20-minute period.

[0064] FIG.22E is a graph depicting pain scores reported by human subjects for the treatments applied in FIG.22D.

[0065] FIG.22F is a graph comparing protein concentrations in ISF from the skin of human subjects normalized to values in plasma from the same human subjects for ISF extracted with different laser pretreatments.

[0066] FIG.22G is a graph comparing protein concentrations in ISF from the skin of human subjects after different laser pretreatments and suction times.

[0067] FIG.23 is a graph comparing ISF collected from the skin of human subjects after application of a cosmetic laser, microneedles, and suction.

[0068] FIG.24 is an optical coherence tomography image of the skin of a human subject after application of 8 pulses of laser, microneedle puncture, and 15 minutes of suction.

[0069] FIG.25A is a graph comparing the effect of later treatment on absolute protein concentration of plasma and ISF from human subjects.

[0070] FIG.25B is a graph comparing the effect of suction time on absolute protein concentration of plasma and ISF from human subjects. Detailed Description

[0071] Improved methods and systems for mobilizing and collecting interstitial fluid (ISF) from the tissue (e.g., skin, mucosal surface, or other tissues) of a subject (e.g., a human, an animal, or another type of living organism) have been developed. The methods and systems described herein may provide improved means for increasing the amount of ISF collected using microneedle-based collection methods. The subject may be referred to herein as a “patient” in need of diagnostic testing that can be carried out on the subject’s ISF, e.g., in place of or as a complement to conventional blood tests. Alternatively, however, the subject may be healthy and the ISF can be collected for any other purpose, including testing of any biomarker that may be present in ISF, such as those for early disease diagnosis, long-term health monitoring, drug 6 53430309.117625-0344 GTRC 2024-159 screening, therapeutic drug monitoring, environmental exposure, physiological status of relevance to athletes, military personnel or others undergoing physical exertion, etc.

[0072] In some embodiments, the methods and systems described herein may be used to sample clinically relevant quantities of ISF during a routine doctor visit, or series of visits. ISF extracted by the methods described herein may have no or minimal contamination from blood, thereby benefitting the accuracy of the biomarker analysis.

[0073] In one aspect, a method is provided that includes (i) applying energy into a selected region of a subject’s skin effective to facilitate ISF mobilization to and / or within the selected region; (ii) inserting an array of microneedles into the subject’s skin at an insertion site to form a plurality of micropores in the subject’s skin in the selected region; and (iii) collecting ISF from the insertion site. The collecting of ISF may include collecting ISF that has flowed from the micropores onto the surface of the subject’s skin or into a fluidic system or reservoir positioned on the subject’s skin at the insertion site. In an alternative process, the collecting of ISF may include collecting ISF via the microneedles inserted at the insertion site. In one embodiment of this process, the microneedles are hollow or porous, and the ISF is drawn into openings (e.g., a lumen, axial bore, or channel defined in the structure of the microneedle) or pores of the microneedle structure. The ISF may be drawn in the microneedle by capillary forces and / or with the aid of a negative pressure (suction) or positive pressure means, such as a syringe or portable vacuum pump or positive pressure apparatus. The microneedle structure may be made of any suitable material, such as a biocompatible metal, ceramic, or polymer. In another embodiment, the microneedles may be made of a hydrogel material, which may be configured following insertion into skin to swell with collected ISF.

[0074] In some embodiments, a total amount of the ISF needed is collected from a single insertion site, and this may occur over a single collection period, which may range from 0.5 minutes to 1 hour, such as 1 minute to 15 minutes. In some other embodiments, multiple collections may occur over two or more different collection times from the single insertion site. In some other embodiments, a total amount of the ISF needed is collected from two or more insertion sites, and those collections may occur in parallel, in series, or in a combination thereof, e.g., at the same or different times.

[0075] In some embodiments, the facilitating of ISF mobilization includes inducing local edema at the selected region of a subject’s skin. The terms “facilitate ISF mobilization” and “increase ISF mobilization” refer to inducing an increase in the rate and / or total amount of ISF that may flow from a micropore created in the skin as compared to the rate and / or total amount of ISF that would flow from the aperture in the absence of the ISF mobilization, such as by 7 53430309.117625-0344 GTRC 2024-159 inducing edema in the skin. In some embodiments of ISF mobilization, skin water content is increased in a way that increases ISF collection. In some embodiments, ISF flow is increased by increasing the driving force for ISF flow and / or by reducing the resistance to ISF flow within the tissue.

[0076] In one particular implementation, the microneedles are inserted into the skin (to puncture the stratum corneum and the viable epidermis, and into the dermis) and then removed right away to make micropores in the subject’s skin. The microneedles may be solid (i.e., non- porous and not hollow) but could be porous or hollow. The punctures could be made at any given site rapidly. It may just be a single application of multiple microneedles, e.g., in a relatively large array, or it may be a smaller array of microneedles or an individual microneedle that is repeatedly inserted into the skin, at exactly or approximately the same area of the skin. That whole process generally may take up to one minute, but would usually be performed in under 10 seconds, for example within 1, 2, 3, 4, or 5 seconds. The process steps might be to apply energy to a target area of the skin, then to apply microneedles into (and possibly out of) the skin in the target area, and then to immediately (or optionally following a delay period) apply suction or positive pressure to the skin. In one scenario, the pressure may cause the ISF to flow out of the pores for collection, e.g., using any suitable collection device, e.g., a cotton swab, or gauze, an absorbent matrix or sponge-like patch, a hydrogel material, a capillary tube, a pipette, or a microfluidic network.

[0077] In another particular implementation, hollow microneedles or porous microneedles (e.g. an array of such microneedles) are inserted into the skin (to puncture the stratum corneum and the viable epidermis, and into the dermis) and then left there for a duration over which ISF is to be collected, which could be up to one week. The ISF may flow through the hollow bores, or the porosities, of the microneedles. The process steps might be to apply energy to a target area of the skin, then to apply the microneedles to the target area, typically immediately after the energy application, and leave them inserted, and then to apply suction or positive pressure to the skin. The suction or pressure, and the collection of ISF, may be carried out immediately (or optionally following a delay period) following microneedle insertion, and may be applied continuously or intermittently to the same area which the microneedles are inserted.

[0078] In some embodiments, the array of microneedles is inserted into the subject’s skin, and then one or more times removed from the subject’s skin and reinserted into the subject’s skin in the selected region. In one approach, the rapid sequential insertions of the array of microneedles may occur within a short interval, e.g., an interval ranging from 1 second to 1 minute, for the purpose of creating many micropores, such as a number of micropores that is 8 53430309.117625-0344 GTRC 2024-159 more than the number microneedles in a single array. For example, the array may be reinserted two, three, or four times to respectively produce two, three, or four times as many micropores as the number of microneedles in the microneedle array.

[0079] In another approach, the rapid sequential insertions of the array of microneedles may occur within a short interval, e.g., an interval ranging from 1 second to 1 minute, for the purpose of re-inserting microneedles into previously created micropores, such that the number of micropores is equal to less than the number microneedles in a single array multiplied by the number of microneedle applications to the skin. This method cannot only generate micropores for ISF flow out of the skin, but can also serve as a mechanical method of introducing energy into the skin that is effective to facilitate ISF mobilization in the skin. For example, the array may be reinserted two, three, or four times to respectively produce fewer than two, three, or four times as many micropores as the number of microneedles in the microneedle array.

[0080] In another approach, a sequential insertion of the array of microneedles occurs following a much longer interval, e.g., an interval ranging from 10 minutes to 24 hours, for the purpose of creating new micropores after at least some of the previously created micropores have closed and / or their ability to pass ISF therethrough has become degraded. The ISF collection methods described herein may use only the first approach, may use only the second approach, or may use a combination of the two approaches together.

[0081] In still another particular implementation, the microneedles inserted into the skin (to puncture the stratum corneum and the viable epidermis, and into the dermis) are hydrogel microneedles or another kind of microneedles that absorbs fluid. The absorption could be into the microneedles themselves and / or into a backing to which the microneedles are attached. The process steps might be to apply energy to a target area of the skin, then to apply the microneedles to the target area, typically immediately after the energy application, and leave them inserted. In this implementation, suction or positive pressure generally would not be used, but it is still be an option to do so.

[0082] In the foregoing specific implementations, the energy may be applied by various suitable means, such as with a laser, and there may be three different time periods to consider in the application of the energy to the subject’s skin in the target area: (1) the duration of an individual application of energy – a single micropulse, or an energy increment; (2) the duration of a set of applications of energy in rapid successful – a train of micropulses, or an energy exposure – referred to as a pulse of energy; and (3) the delay between applications of pulses of energy, such as in multiple energy exposures. The purpose of #2 is generally to heat or otherwise damage the skin to cause edema. However, the purpose of #1 is generally to regulate 9 53430309.117625-0344 GTRC 2024-159 the amount of heat and how it dissipates in the skin so that one does not cause too much injury. The purpose of #3 is to repeat what was done in #2 because the effects have worn off and / or one needs to make more edema or make edema again.

[0083] There are various time constants to consider in the process. For example, there is the duration of each micropulse of energy. Non-limiting examples of such micropulse durations are 5 to 50 ms, such as about 15 ms. There is the duration of each pulse of energy which could be comprised of multiple micropulses, which may vary depending on the energy delivery method. Non-limiting examples of such durations are 0.1 to 10 s for laser, 1 s to 1 min. for RF, and 1 ms to 10 mins for ultrasound. There may be a delay between repeated application of energy pulses. Non-limiting examples of such durations are 1 µs to 10 mins, such as between 1 s and 5 s. There also is the duration over which microneedles are rapidly applied repeatedly in and out of the skin. Non-limiting examples of such durations are 1 ms to 10 s (for each application (single insertion and removal)), taking 1 second to 1 minute for a set of applications. There is the duration over which microneedles are pressed into the skin and left there. (Non-limiting examples of such durations, such as for hollow or hydrogel microneedles, are up to 1 week.) There is the delay between sets of applications of microneedles. (Non-limiting examples of such durations are 10 minutes to 24 hours.) There also is the delay between applying microneedles and applying suction or pressure (e.g., up to 12 hours or 1 day), and there is the delay between repeated applications of suction or pressure (e.g., up to 1 day).

[0084] The methods for collecting ISF disclosed herein may include applying a laser pretreatment to the skin to subject's skin to increase water content, applying an array of microneedles to the skin to provide pathways for ISF flow out of the skin, and collecting ISF via suction According to a preferred embodiment, a method for collecting ISF from a subject's skin includes depositing energy into a treatment area on the subject's skin, via a laser, to facilitate ISF mobilization within the treatment area; inserting an array of microneedles into the treatment area to form a plurality of micropores in the subject's skin at the insertion site; and collecting ISF, via suction, that flows from the micropores to the surface of the subject's skin.

[0085] The deposition of energy and the application of the microneedles may be conducted in any suitable order. For example, the microneedles may be applied to the subject's skin prior to deposition of energy into the tissue, or following deposition of energy into the tissue. ISF may be collected before and / or after the application of energy (although preferably after application of energy in most circumstances), following application of the microneedles to the tissue. 10 53430309.117625-0344 GTRC 2024-159

[0086] In some embodiments, the methods described herein enable extraction of more than 5 µl of ISF from a subject's skin, or more preferably, more than about 10 µl of ISF, more than about 15 µl of ISF, more than about 20 µl of ISF, more than about 25 µl of ISF, more than about 30 µl of ISF, more than about 40 µl of ISF, more than about 50 µl of ISF, or more than about 100 µl of ISF. In some embodiments, the methods enable collection of about 0.1 µl / minute to about 100 µl / minute, such as between 1 µl / minute and 20 µl / minute. ISF may be collected over a total period of 0.5 minutes to 1 hour, such as 1 minute to 15 minutes.

[0087] The methods of collecting ISF described herein may facilitate ISF mobilization within the tissue. Inducing local edema at the collection site has been demonstrated as the mechanism effective to facilitate ISF mobilization by application of energy to the skin.

[0088] In some embodiments, inducing local edema is effected by selective application of energy to the subject's skin at or about the insertion site. Application of energy to the subject's skin may increase ISF mobilization within the epidermis, at the dermo-epidermal junction, and / or in the dermis. In some embodiments, application of energy to the subject's skin may generate heat within the tissue and / or on the tissue surface, which may be effective to mobilize ISF within the skin and / or induce local edema within the skin.

[0089] In some embodiments, energy is applied to the skin continuously for a selected period of time. In some other embodiments, energy is applied repeatedly with intermittent periods of less or no energy being applied. For example, energy may be applied, continuously or intermittently, over a period of from 1 microsecond to 10 minutes. The time period over which energy is applied may be determined at least in part by the amount of time necessary to mobilize ISF in a manner sufficient to produce the desired amount of ISF to be collected. In some embodiments, the amount of time necessary to mobilize ISF in a manner sufficient to produce the desired amount of ISF to be collected is the amount of time needed to induce visible edema in the tissue.

[0090] The energy may be applied to a selected area or region of the subject's skin. In some embodiments, energy may be applied only at a single location in the tissue region. In other embodiments, energy may be applied to multiple locations in the tissue region. The multiple locations may be spaced apart from one another or in an overlapping fashion. In some embodiments, the selected tissue region is between about 0.1 cm2and 100 cm2.

[0091] In some embodiments, energy is applied to the tissue only once. In other embodiments, energy is applied to the tissue in multiple successive depositions. For example, energy may be applied to the tissue up to 100 times over the period of 1 day. In some embodiments, the delay between energy applications may be 1 minute, 2 minutes, 5 minutes, 10 11 53430309.117625-0344 GTRC 2024-159 minutes, 30 minutes, 1 hour, 2 hours 5, hours, 6 hours, 12 hours, or 1 day. Other, shorter or longer, intervals of no energy application are envisioned.

[0092] In some embodiments, inducing local edema is effected by selective application of electromagnetic energy to the subject's skin at or about the microneedle insertion site. The electromagnetic energy may be in the form of one or more electric pulses applied with at least two electrodes, or in the form of visible light or ultraviolet radiation (e.g., UVA, UVB, UVC light). In some embodiments, local edema is effected by treating the skin with ultraviolet, visible, near infrared, or infrared light, which may be continuous or pulsed, and may be applied by a laser.

[0093] In some embodiments, the local edema is effected by application of a bioactive agent onto and / or into the subject's skin or about the selected skin area, to increase the local vasodilation, to increase local vascular permeability, to increase interstitial oncotic pressure, to decrease plasma oncotic pressure, or a combination thereof.

[0094] In some embodiments, inducing local edema is effected with radiofrequency (RF), including a fractional treatment of the skin using, for example, monopolar, bipolar, or multipolar RF energy application. While RF treatment is often used for cosmetic procedures, it can also be used to generate inflammation in the skin. For example, RF energy can be applied for about 10 seconds to about 5 minutes, such as for about 30 seconds to about 3 minutes, or about 1 minute to about 2 minutes, at a frequency of about 0.1 MHz to about 10 MHz, such as about 0.5 MHz to about 2 MHz, or about 1 MHz using energy settings typical of those used for RF treatment for skin tightening. In some embodiments, the power of the RF energy is between about 0.1 Watts and about 10 Watts, such as between about 0.5 Watts and about 5 Watts, or around 1 Watt.

[0095] In some embodiments, the RF device may be used to continuously apply energy to the skin for a period of 1 second to 120 seconds, such as between 5 seconds and 60 seconds, or between 10 seconds and 30 seconds. In some embodiments, RF energy is applied to the tissue intermittently, with each application being separated by an interval between 1 second and 120 seconds, such as between 5 seconds and 60 seconds, or 10 seconds and 30 seconds.

[0096] In some embodiments, a device with output RF energy may be used. The nominal power output of some devices may be about 5 W. In some embodiments of the methods described herein, the energy may be applied to the skin as RF energy having a frequency ranging from around 0.1 MHz to around 10 MHz, such as from 1 MHz to 6 MHz, and delivery of the energy input may occur over a period from 1 second to 5 minutes. The delivery of RF energy may be continuous or intermittent. The power per surface area of the skin treated may range from 0.05 W / cm2to around 10 W / cm2. 12 53430309.117625-0344 GTRC 2024-159

[0097] In some embodiments, a device with output ultrasound energy frequency of approximately 0.02 MHz to about 5 MHz may be used. The ultrasound energy power may range from 0.1 W / cm2to 10 W / cm2. The delivery of ultrasound energy may be continuous or intermittent.

[0098] In some embodiments, a device providing AC or DC current directly to the skin may be used. Such devices known in the art may be adapted for use to deliver energy into the skin according to the present methods.

[0099] In some embodiments, local edema is effected by direct application of heat, such as by conduction from a heating apparatus placed in contact with the target skin area.

[0100] In some embodiments, local edema is effected by mechanical means. Examples include massaging (e.g., manual manipulation of the skin) or vibration with continuous or discontinuous (e.g., tapping) contact of a vibration device with the skin, with movements in the x, y and / or z directions. The mechanical intervention may be non-penetrating (applying forces to the skin surface) and / or penetrating (something punctures or otherwise enters the skin). The microneedles themselves may serve a dual function of a mechanical means of making edema and to create pores for ISF flow.

[0101] In the various methods of applying energy to the skin described herein, the local heating of a selected area of the skin may be to a temperature of about 40 °C to about 60 °C, about 40 °C to about 55 °C, about 40 °C to about 50 °C, about 45 °C to about 60 °C, about 45 °C to about 55 °C, about 45 °C to about 50 °C, about 50 °C to about 60 °C, or about 50 °C to about 55 °C.

[0102] In a preferred embodiment, inducing local edema is effected with a laser, which may be used to achieve an ablative or non-ablative, fractional or non-fractional laser treatment of the skin. While these laser treatments are often used for cosmetic procedures, they can also be used to generate inflammation in the skin. In some embodiments, a subject's skin is treated with laser before application of the array of microneedles and / or collection of ISF from the skin.

[0103] In some embodiments, the laser has a wavelength between 950 nm and 1500 nm, preferably between 950 nm and 1000 nm. In some embodiments, the laser has a wavelength between 1050 nm and 1200 nm. In some embodiments, the laser has a wavelength between 1350 nm and 1500 nm. In some embodiments, the laser has a wavelength between 1800 nm and 2100 nm. In some embodiments, the laser has a wavelength between 300 nm and 400 nm, or between 500 and 600 nm.

[0104] In some embodiments, each laser pulse has a duration between 0.1 ms and 30 ms, such as between 2 ms and 20 ms, between 5 ms and 15 ms. In some embodiments, each laser 13 53430309.117625-0344 GTRC 2024-159 activation has a duration between 0.1 s to 10 s, such as between 0.5 s to 5 s, or 1 s. As used herein, "laser activation" refers to the period between the user's activation of the laser and the time the laser deactivates, during which time laser energy may be emitted continuously or intermittently. The laser activation period may also be referred to as a "pulse" or an "energy exposure" and the pulse, i.e., the time the laser is actually depositing energy, may be referred to as a "micropulse" or an "energy increment".

[0105] In some embodiments, the laser will emit 1 to 100 micropulses for each pulse or activation at a predetermined frequency. In some embodiments, the laser micropulse frequency is from 1 Hz to 30 Hz, such as from 5 Hz to 25 Hz, or 10 Hz to 20 Hz. In some embodiments, the energy output per pulse is between 5 mJ and 300 mJ, or more preferably, between 50 mJ and 150 mJ.

[0106] The energy exposure (e.g., from the laser) per surface area of skin may vary. In some embodiments, this value is between 0.1 J / cm2and 20 J / cm2, such as between 1 J / cm2and 6 J / cm2including only the region treated by each micropulse, or between 0.1 J / cm2and 0.3 J / cm2including the treated and untreated regions there between.

[0107] The methods of collecting ISF described herein preferably include increasing ISF flow and / or mobility within the dermal tissue. In some embodiments, insertion into the skin of an array of microneedles (once, or optionally two, three, four, or more times) is effective to improve ISF mobilization. Therefore, the plurality of microneedles may be inserted and removed from the insertion site of the skin several times prior to collecting ISF.

[0108] In some embodiments, puncturing the skin more than once with a plurality of microneedles creates a plurality of micropores very close to each other (e.g., within a few millimeters of each other, about 1mm from each other, less than 1 mm from each other, or at the same location, where microneedle insertions puncture the skin at skin areas that at least partially overlap with skin areas that were previously punctured). In some embodiments, the plurality of microneedles may be inserted into the subject's skin in a manner that produces two or more regions of micropores that are very close to each other. The regions may be one or more centimeters apart. The closely spaced punctures may, in some instances, further create local injury or irritation that facilitates ISF flow or creation at the site of the repeated microneedle puncture.

[0109] The array of microneedles may be an array of microneedles configured for insertion across the stratum corneum of a subject's skin, each microneedle having an outer surface, an insertion tip end portion, and a base portion, and a backing structure from which the array of microneedles extend. The microneedles preferably are solid, i.e., have no hollow bore. In other 14 53430309.117625-0344 GTRC 2024-159 suitable embodiments, the microneedles are hollow, i.e., include a hollow bore extending axially therethrough or the microneedles are porous, i.e., include multiple pores forming a network of pores in the microneedle.

[0110] Each microneedle has a proximal base portion attached, directly or indirectly, to the backing structure, and a distal tip end portion. The tip end portion has a shape, dimensions, and rigidity effective to penetrate the stratum corneum of skin, such as human skin. In some cases, the microneedles may be dimensioned and shaped to avoid or minimize puncturing of blood vessels within the skin. The skin may be the skin of a human or other mammal, animal, plant or other living organism. It is envisioned that the present devices and methods may also be adapted to other biological tissues and other animals.

[0111] In some embodiments, the microneedles may have tapered sidewalls between the proximal and distal ends. The tapered sidewall may extend all or a portion of the distance between the proximal and distal ends of the microneedles.

[0112] In some embodiments, the length of a microneedle (LMN) is between about 50 µm and 2 mm. In some preferred embodiments, the LMN is between 200 µm and about 1200 µm,and ideally between about 250 µm and about 750 µm, or µm to about 350 µm or about 600 µm to about 750 µm. In one embodiment, the array of microneedles includes from 10 to 1000 microneedles. In some embodiments, the microneedle tips have a radius of curvature of 1 µm to 100 µm, in which the radius of curvature is indicative of the sharpness of the microneedle tip.

[0113] In some embodiments, the microneedles are water-soluble, i.e., are designed to at least partially dissolve following insertion into a biological tissue or organ, e.g., the skin. For example, such microneedles may be made of a water-soluble polymer (such as polyvinyl alcohol), a sugar, or a combination thereof. This may be desirable so that the microneedles cannot be inadvertently or intentionally re-inserted into skin following its use with a first subject.

[0114] In some other embodiments, the microneedles are not water-soluble, i.e., are designed not to dissolve in the tissue. For example, such microneedles may be made of a metal (such as stainless steel or titanium), a ceramic, or a biodegradable or a nonbiodegradable polymer. Examples of suitable polymers include cross-linked polymers, such as gelatin methacryloyl, or non-cross-linked polymer, such as polylactic acid or poly(lactic-co-glycolic acid), or a combination thereof.

[0115] In some embodiments, application of the array of microneedles to a tissue site (e.g., skin tissue) is effective to create between 2 and 10,000 micropores in the tissue. The array of 15 53430309.117625-0344 GTRC 2024-159 microneedles may be applied to the tissue a single time, or may be inserted, removed, and re- inserted to form the optimal number of micropores in the tissue. In various embodiments, the number of micropores created by application of a microneedle to a selected tissue site (e.g., skin) may range from 1 to 1000 micropores, such as between 10 and 500 micropores, between 50 and 500 micropores, or between 100 and 300 micropores in the tissue.

[0116] In some embodiments, the micropores are formed by inserting the array of microneedles into the subject's skin, removing the array of microneedles, and reinserting the array of microneedles. Just like for the repeated energy exposures, the microneedle applications may be comprised of multiple rapidly administered microneedle punctures, and there may also be multiple microneedle applications with wider separations between applications. In one scenario, the microneedles are rapidly pressed into and out of the skin in a manner effective to create many micropores, using a microneedle array that has a relatively small number of microneedles (e.g., 2 to 20). The delay between insertions could range from 1 ms to 10 s. All of these insertions occur in a short time frame, e.g., 10 s to 1 minute. In another scenario, the microneedles are pressed into the skin (possibly repeatedly) to create micropores, then, following a longer delay, the microneedles are reapplied (possibly repeatedly) to create new pores since the old pores are closing or are closed or are otherwise of reduced functionality to permit ISF flow. Here, the delay between microneedle applications to the skin may be from 10 min and 24 hours. The interval between the sequential insertions of the array of microneedles may range from between 0.1 seconds to 24 hours, or more preferably, between 1 second and 5 minutes. During each insertion, the microneedles may remain in the skin for a period of 0.1 seconds to 24 hours, but preferably are removed within 0.1 seconds to 3 seconds of insertion.

[0117] In some embodiments, ISF collection begins while the microneedles remain in the tissue, such that ISF flows in the space around the microneedle in the micropore to facilitate collection. In other embodiments, ISF collection does not begin until after the microneedles have been removed from the tissue.

[0118] ISF collection may be facilitated by application of a positive or negative pressure gradient to the tissue. In some embodiments, the pressure gradient is a positive pressure gradient, with the peak positive pressure being from 10 kPa to 1500 kPa. According to a preferred embodiment, ISF collection is facilitated by the application of suction (i.e., a negative pressure gradient) to the tissue. In some embodiments, the peak negative pressure is between -5 kPa and -100 kPa, preferably between -30 kPa and -80 kPa.

[0119] In some embodiments, the positive or negative pressure is applied to the periphery of the tissue in the collection area, i.e., the area to which energy and microneedles have been 16 53430309.117625-0344 GTRC 2024-159 applied. In other embodiments, the positive or negative pressure is applied to all the tissue in the treatment area to facilitate flow of ISF through the micropores in the tissue. In further embodiments, the positive or negative pressure is applied to the periphery of the pores or micropores of the tissue in the treatment area, or is applied adjacent to the pores or micropores of the tissue in the treatment area.

[0120] In some embodiments, the positive or negative pressure is applied to collect ISF over a duration of between 1 minute and 1 week, such as 2 minutes, 5 minutes, 10 minutes, 20 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 5 hours, 6 hours, 12 hours, or 1 day. For example, suction may be repeatedly applied at intermittent intervals over a period of up to 1 week after the initial treatment of the tissue with energy deposition and / or microneedles. The gap between intermittent applications of suction may be between 1 minute and 1 day, such as 2 minutes, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 5 hours, 6 hours, 12 hours, or 1 day. In some embodiments, application of suction may be delayed after the initial treatment of the tissue with energy deposition and / or microneedles for a period of 1 minute to 1 day, such as 2 minutes, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 5 hours, 6 hours, or 12 hours. In some embodiments, the skin may be cleaned between application of energy and microneedles to the tissue and application of suction, which may improve ISF collection.

[0121] The ISF collection process may include application of energy, plus application of the microneedle array, plus application of positive or negative pressure to the skin tissue (energy+microneedle+pressure), each time an ISF collection is performed. However, it has been discovered that in one approach a first collection uses energy+microneedle+pressure while one or more subsequent collections use only energy+pressure or only microneedle+pressure or only pressure. It was surprisingly discovered that, after applying energy+microneedle+pressure to a skin area at an initial time, ISF could be collected from that site up to at least 7 hours after the initial energy+microneedle treatment, and without needing to reapply the energy or the microneedles.

[0122] In some embodiments, the application of a pressure gradient includes repeated intermittent application of positive or negative pressure, such as at a frequency ranging from about 0.2 Hz to about 30 Hz. In this way, the subject’s skin (around the micropores or microneedles) may be caused to move, or vibrate, in a manner that pushes ISF toward the micropores, allows the skin to recover, and then pushes more ISF, effectively pumping ISF toward / through / into the micropores or microneedles. 17 53430309.117625-0344 GTRC 2024-159

[0123] Collection of ISF that flows from the micropores to the surface of the subject’s skin may be carried out immediately or nearly immediately following the steps of applying energy and inserting the array of microneedles into the skin. Alternatively or in addition, the collection of ISF that flows from the micropores to the surface of the subject’s skin may be carried out following a delay period after the steps of applying energy and the inserting the array of microneedles. The delay period may be useful to allow time for additional ISF within the tissue to flow toward the micropores.

[0124] The methods described may, and typically do, further include analyzing the collected ISF for the presence or concentration of one or more substances. The one or more substances may include a biomarker indicative of a specific disease, injury, environmental exposure, or physiological status. In some embodiments, the one or more substances include a pharmaceutical compound or metabolite.

[0125] The methods described herein may be used for disease detection and / or monitoring in humans or other mammals. In some embodiments, the methods include collecting ISF from a biological tissue; and analyzing collected ISF for the presence or concentration of (i) a biomarker indicative of a specific disease, injury, environmental exposure, or physiological status, (ii) a pharmaceutical compound, (iii) a metabolite, or (iv) a combination thereof; wherein collecting ISF includes applying a laser to the biological tissue to facilitate ISF mobilization within the tissue; applying an array of microneedles to the biological tissue to form a plurality of micropores in the biological tissue to facilitate ISF collection; and collecting, via suction, ISF that flows from the micropores in the biological tissue. The biological tissue generally may be any human or animal tissue that is an internal or external tissue. In some embodiments, the biological tissue is mammalian skin, particularly human skin.

[0126] In some embodiments, the methods described herein are used to detect or determine an amount of troponin I in ISF. Troponin I is a sensitive marker of myocardial injury, and is necessary for establishing the diagnosis of myocardial infarction (MI). In some embodiments, the methods described herein include applying a laser to the biological tissue to facilitate ISF mobilization within the tissue, and applying an array of microneedles to the biological tissue to form a plurality of micropores in the biological tissue to facilitate ISF collection of a subject showing symptoms of MI, collecting ISF, via suction, and contacting the ISF with a fibrous, porous, or otherwise absorbent matrix functionalized to selectively bind with troponin I, and analyzing the ISF (e.g., by a spectroscopic measurement, a colorimetric detection method, or other method) to determine an increase in troponin I in ISF. 18 53430309.117625-0344 GTRC 2024-159

[0127] In some embodiments, the methods described herein are used to detect infectious diseases, such as COVID or influenza or malaria. In some embodiments, the methods described herein include applying a laser to the biological tissue to facilitate ISF mobilization within the tissue, and applying an array of microneedles to the biological tissue to form a plurality of micropores in the biological tissue to facilitate ISF collection of a subject, collecting ISF, via suction or positive pressure, and contacting the ISF with a fibrous, porous, or otherwise adsorbent matrix, possibly functionalized with antibodies specific to the antigen, and analyzing the ISF (e.g., with a paper based enzymatic assay, colorimetric transduction method, or other method) to determine the presence and / or concentration of antibodies or antigens to diagnose the infectious disease.

[0128] In some embodiments, the methods described herein are used to detect exposure to one or more hazardous substances, including those commonly associated with terrorism or chemical warfare. In some embodiments, the methods described herein include applying a laser to the biological tissue to facilitate ISF mobilization within the tissue, and applying an array of microneedles to the biological tissue to form a plurality of micropores in the biological tissue to facilitate ISF collection of a subject, collecting ISF, via suction or positive pressure, and contacting the ISF with a fibrous, porous, or otherwise adsorbent matrix functionalized with nanoparticles, and analyzing the ISF (e.g., with a Surface Enhanced Raman Spectroscopic method, or other method) to determine the presence of the hazardous substance.

[0129] In some embodiments, the methods and / or devices described herein are used to monitor, optionally continuously, concentrations of drugs and / or biomarkers in ISF, including lactate, cortisol, vitamin D, and vitamin E. For example, as described herein, the methods and / or devices may be used for therapeutic drug monitoring.

[0130] The methods and systems described herein may have a number of commercial applications, including, but not limited to, the discovery and / or validation of novel biomarkers, the monitoring of concentrations of drugs and / or biomarkers in ISF during drug development, continuous monitoring of biomarkers, therapeutic drug monitoring, or a combination thereof.

[0131] As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. The term "about", as used herein, indicates the value of a given quantity can include quantities ranging within 10% of the stated value, or optionally within 5% of the value, or in some embodiments within 1% of the value.

[0132] Various numerical ranges may be disclosed herein. When Applicant discloses or claims a range of any type, Applicant's intent is to disclose or claim individually each possible 19 53430309.117625-0344 GTRC 2024-159 number that such a range could reasonably encompass, including end points of the range as well as any sub-ranges and combinations of sub-ranges encompassed therein, unless otherwise specified. EXAMPLES

[0133] The invention can be further understood with reference to the following non-limiting examples, which are not to be construed in any way as imposing limitations upon the scope thereof. On the contrary, it is to be clearly understood that resort may be had to various other aspects, embodiments, modifications, and equivalents thereof which, after reading the description herein, may suggest themselves to one of ordinary skill in the art without departing from the spirit of the present invention or the scope of the appended claims. Thus, other aspects of the invention will be apparent to those killed in the art from consideration of the specification and practice of the invention as disclosed herein. Example 1: Microneedle Array Fabrication

[0134] Microneedles were designed in SOLIDWORKS (Dassault Systemes, Vélizy- Villacoublay, France) and fabricated from grade 304 stainless steel sheets by photo etching (Tech-Etch, Plymouth, MA and Great Lakes Engineering, North Maple Grove, MN). Microneedles were fabricated as 5-linear arrays (i.e., microneedle arrays) with 1250 µm tip-to- tip spacing, a thickness of 50 µm, a base width of 200 µm at the array backing, and tapering to a sharp tip with radius of 10 µm. For experiments ex-vivo, microneedles were 750 µm long. For in vivo experiments, microneedles were 250 µm long. Example 2: Animal Study Procedures

[0135] All animal study procedures were approved by the Georgia Institute of Technology Institutional Animal Care and Use Committee (IACUC). Eight male Lewis rats and four female Wistar rats age 15-30 weeks-old (Charles River Laboratories, Wilmington, MA) were anesthetized using isoflurane (AErrane, Baxter Healthcare, Deerfield, IL) inhalation, and hair from both lateral flanks was removed by shaving and application of depilatory cream (Nair, Princeton, NJ), which was wiped clean with gauze and deionized water at least 24 hours prior to laser treatment.

[0136] The day of laser treatment, rats were anesthetized again using isoflurane, and eight laser pulses were applied within a 2.3 cm2area of one side of the rats. After 5 min from the first laser pulse application, an 8 mm skin biopsy was taken from the treated area, blood gently removed by blotting, and weighted, and the mass was recorded. A biopsy from the untreated site of the same rat was taken as a control. The order of the control and treated biopsy was alternated 20 53430309.117625-0344 GTRC 2024-159 between rats to control potential sampling order effects. After the mass of the biopsies were recorded, the rats were euthanized by asphyxiation using carbon dioxide.

[0137] The biopsies were frozen at -20 °C, and the frozen biopsies were lyophilized for 24 hours to remove over 95% of the water in a Labconco Freezone 6-liter freeze dryer (Kansas City) at -50 °C and less than 0.1 mbar. The dry biopsy mass was recorded, and the water content was calculated by assuming all the mass removed by lyophilization was water. Example 3: Measurement of ISF Extracted from Skin Ex-Vivo as a Function of Pressure

[0138] Interstitial fluid (ISF) was extracted ex-vivo from pig skin from the inner pinna of the ear at various pressures using suction or positive pressure. After removing subcutaneous fat, skin samples were cut into 24-mm diameter pieces and incubated for about 20 hours in a Franz cell with the receiver chamber containing 1 mg / ml fluorescein in phosphate buffer saline (PBS). In this way, the skin ISF was loaded with fluorescein to facilitate subsequent measurements. The skin was then removed from the Franz cell and immediately stretched on a stage using pins and treated with microneedles by pressing and immediately removing a microneedle array with five 750 µm long microneedles twice into the skin, creating 10 micropores.

[0139] For experiments using suction, suction was applied for 5 minutes using a polyvinyl chloride hose with the inner diameter of 10 mm placed on the surface of the skin surrounding the sites of microneedle puncture. A pressure regulator on the suction line from a vacuum pump (KNF Neuberger, Freiburg, Germany) was used to adjust the suction to the desired pressure of - 10, -25, -50 or -75 kPa. In some cases, suction applied with a cosmetic pump (VOYOR BR610 Blackhead Remover, Hymax, London, United Kingdom) coupled to a plastic adapter included by the equipment manufacturer. In all suction experiments, suction pressure was applied uniformly across the skin area under the hose or plastic adapter (FIG.1A). This mobilized ISF in the skin immediately next to each microneedle pore.

[0140] For experiments using positive pressure, a hollow cylinder made of glass with an inner diameter of 9.0 mm and an outer diameter of 12.5 mm was placed on top of the skin, which created a contact area of 0.64 cm2between the cylinder and skin. Pressure was applied for 5 min by placing a mass on top of the cylinder. Masses of 4.8, 2.4, 1.2, 0.6, 0.3, 0.15, and 0.06 kg were used to apply pressures of 800, 400, 200, 100, 50, 25, and 10 kPa, respectively. This pressure was applied only to the skin in contact with the cylinder, forming a ring of pressure surrounding the skin area with microneedle punctures (FIG.1B). The site of pressure application to the skin was of variable distance from each individual microneedle pore, less effectively mobilizing ISF flow in the skin adjacent to each microneedle pore. 21 53430309.117625-0344 GTRC 2024-159

[0141] The hydraulic conductivity of the dermis was calculated based on a model previously developed for scleral tissue in the eye. Like the dermis, the sclera is a connective tissue that is rich in collagen and glycosaminoglycans (GAGs).

[0142] Net hydraulic conductivity of dermis (Kdermis) was calculated by modeling the dermis as a composite medium in which two distinct domains for fluid flow are available. The first domain (the microscale) consists of pores formed by randomly oriented GAG and proteoglycan (PGP) fibers in the dermis. The second domain (the mesoscale) consists of a medium in which collagen fibers are assumed to be periodically arranged (FIG.2).

[0143] In both sets of experiments (i.e., experiments using suction and positive pressure), the fluid on the surface of the skin was wiped off using a Kimwipe (Kimberly-Clark, Irving, TX) cut into pieces measuring approximately 25 mm by 25 mm. Fluorescein from the fluid extracted from the skin was eluted from the Kimwipe by incubating overnight in DI water at room temperature (20 °C-25 °C), and the total amount of fluorescein in the ISF was quantified by fluorescence using a Biotek Synergy plate reader (Agilent Technologies, Santa Clara, CA).

[0144] ISF volume was calculated by multiplying the mass of fluorescein collected by the concentration of fluorescein in which the skin was incubated. This analysis assumes that the ISF in the skin had the same fluorescein concentration as in the receiving chamber of the Franz cell in which it was incubated. This assumption was validated by extracting ISF in a 5 ml capillary, measuring the volume of fluid by the height of the capillary column, and quantifying the fluorescence in the known volume of fluid. Example 4: Measurement of ISF Extracted from Skin Ex-Vivo After Enzyme Treatment

[0145] Collagenase types I (250 U / mg activity) and III (≥ 100 U / mg) were obtained from MilliporeSigma (Burlington, MA); collagenase type V (≥450 U / mg) was obtained from Worthington Biochemical (Lakewood, NJ); and hyaluronidases types I (700 U / mg) and II (300 U / mg) were obtained from MilliporeSigma (Burlington, MA). In experiments that varied enzyme concentration, only collagenase type I was used.

[0146] To test the effect of different enzymes on ISF extraction, the procedure described above was used, except the Franz cell receptor solution contained one or more enzymes at a concentration of 0.5-30 mg / ml in addition to PBS and fluorescein. Formulations of hyaluronidase as low as 100 U / ml have been shown to reduce the pressure necessary for injection 1 ml of fluid into the dermis, and therefore enzyme concentrations near or above this threshold were chosen. The volume of ISF extracted from treated samples was compared to the volume extracted from 5 to 9 untreated samples incubated without enzyme in the same set of experiments. 22 53430309.117625-0344 GTRC 2024-159 Example 5: Measurement of ISF Extracted from Skin Ex-Vivo at Different Skin Hydrations

[0147] To extract ISF from skin with different water contents, a modified version of the procedure for ISF extraction at different pressures was used, in which skin water content was modified after the Franz cell incubation and prior to ISF extraction.

[0148] After Franz cell incubation to fully hydrate the skin, the skin mass was recorded. To change skin water content, the skin was placed on a piece of Tork paper towel (Essity Professional Hygiene, Philadelphia, PA) dermis-side down, and a mass of 8.9 kg was placed on top of the skin (i.e., stratum corneum side) to drive fluid out of the skin by convection. Compressing the skin for different durations (0.5 to 20 minutes) resulted in different average levels of skin water content (66.9 ± 3.0% to 57.7 ± 4.3% water). After removing the 8.9 kg mass, the mass of the skin was recorded again, and ISF was extracted from 10 micropores in the skin created by pressing 750 µm-long microneedles, as described above.

[0149] After extracting ISF (mISF), the active area of the skin (i.e., the area of skin exposed to fluid in the Franz cell) was collected using an 8-mm biopsy (Integra Lifesciences, Princeton, NJ), its mass was recorded (mskin), and then the skin was lyophilized for 24 hours to remove over 95% of the water in a Labconco Freezone 6-liter freeze dryer (Kansas City, MO) at -50 °C and less than 0.1 mbar. Skin mass was recorded (mlyo), and the mass loss during lyophilization (mwater) was assumed to be all water. The hydration of the skin (W) was calculated using the following equation: ^^^^^^ െ ^^^^ ൌ ^௬^^^,^^^^^^ ൌ ^^^௬^ ^ ^^ூௌி ^ ^^௪^௧^^,^^^^^^^^^^^^^^^^ 1^^^^where mskin,water loss during lyophilization, and ISF extracted from skin, respectively. Example 6: Histology of Collagenase-Treated Dermis

[0150] To evaluate the effect of collagenase on the skin, histological analyses were completed using hematoxylin and eosin (H&E) staining of skin samples treated with collagenase for 20 hours. To prepare the skin samples, pig ear skin was incubated in Franz cells filled with PBS and collagenase (10 mg / ml) in the receiver chamber for 20 hours. A 6-mm biopsy was taken from the skin, which was then sectioned into 5-10 µm slides, collected on microscope slides, fixed and stained with H&E. A coverslip was added to the slide and the tissue was visualized by optical microscopy (Olympus BX41, Evident Corporation, Tokyo, Japan). 23 53430309.117625-0344 GTRC 2024-159 Example 7: Measurement of Effective Hydraulic Conductivity of Dermis Ex-Vivo

[0151] To measure effective hydraulic conductivity of dermis, an apparatus was built to measure the volume of fluid flowing through the dermis under controlled transdermal pressure at known time intervals but designed to operate at larger pressures. Because tissues deform at large pressures and the area, thickness, and microstructure may differ from the undeformed tissue, the hydraulic conductivity measured may differ from the hydraulic conductivity of the tissue without deformation. Therefore, these measurements are referred to as “effective hydraulic conductivity.”

[0152] Hydraulic conductivity of the dermis was measured using the sampling apparatus 100 shown in FIG.3, in which a piece of dermis S was sandwiched between two acrylic plates 102, 104. Between the dermis and the plates were a bottom O-ring 106 (12.5 mm inner diameter, 2.5 mm thickness) and a top O-ring 108 (10 mm inner diameter, 3 mm thickness). The bottom plate 102 had a 9 mm hole to expose the dermis to pressurized PBS supplied through supply line 120 connected to adaptor 122. The top plate 104 had a 9 mm hole that served as a cylindrical sampling chamber 112 exposed to air (i.e., at atmospheric pressure). To reduce strain on the dermis produced by tissue deformation from the transdermal pressure gradient, a 3D printed polymer mesh 110 was placed on the upper side of the dermis. Bolts 116 and nuts 118 secured the plates together.

[0153] Three of these sampling apparatuses 100 were assembled as part of a larger apparatus 200 to apply a controlled pressure gradient across the skin as shown in FIG.4.

[0154] The three sampling apparatuses 100 were connected in parallel via polyvinyl chloride tubing 202. In addition to the three stages, there was a pressure gauge 204 at the end of the line which read the pressure drop after all three T-junctions 206 leading to the sampling stages.

[0155] Pressurized phosphate buffer saline (PBS) was supplied to the sampling stages by a PBS reservoir 210 which was pressurized by air A from an air compressor. An air pressure regulator 212 allowed to set the pressure, via regulator knob 214, of the air in contact with the PBS in the reservoir. The pressure drop from between the air regulator and the pressure gauge at the end of the line was less than 0.5 psi. The total pressure drop across the full system (i.e., from the first pressure gauge to the second pressure gauge) was less than 0.5 psi. This pressure drop included the cumulative pressure drop across all three T-junctions leading to the sampling chambers. The pressure of the samples in the stage was assumed to be the pressure read by the gauge at the end of the line.

[0156] The volume of fluid flowing across the skin and collected during the hydraulic conductivity measurements at different pressures shows an increase in pressure. Notably, 24 53430309.117625-0344 GTRC 2024-159 volumes collected at 200 kPa were larger than expected for constant hydraulic conductivity (FIGS.5-6). Note that the flow rate across dermis increased 20-fold over this six-fold increase in pressure, which cannot be explained just by an increased convective driving force, which increases in direct proportion to the pressure, and therefore requires a change in hydraulic conductivity.

[0157] To measure the effective hydraulic conductivity in the apparatus, PBS was used as the fluid flowing through the tissue. PBS is virtually isotonic with extracellular fluid and ISF, and therefore using PBS as the fluid flowing through the dermis reduced the potential to create osmotic pressure gradients that could alter the net volume of ISF crossing through the dermis (or skin) due to the mechanical pressure differential.

[0158] Pig ear skin was cut into circles with diameter of 30 mm and weighed. The epidermis was removed by first loosening the dermoepidermal junction by incubation in a water bath at 55 °C for 90 seconds followed by placement in a room temperature water bath for 5-10 minutes. Then, the epidermis was scraped off using a metal spatula, leaving the dermis behind, which was weighed and mounted in a hydraulic conductivity measurement apparatus.

[0159] In the apparatus, a piece of dermis was sandwiched between two acrylic plates. Between the dermis and the plates were a bottom O-ring (12.5 mm inner diameter, 2.5 mm thickness) and a top O-ring (10 mm inner diameter, 3 mm thickness). The bottom plate had a 9 mm hole to expose the dermis to pressurized PBS, and the top plate had a 9 mm hole that served as a cylindrical sampling chamber exposed to air (i.e., at atmospheric pressure). To reduce strain on the dermis produced by tissue deformation from the transdermal pressure gradient, a 3-D printed polymer mesh was placed on the upper side of the dermis.

[0160] To verify dermal integrity, a pressure of 3 kPa was applied to the dermis, and samples which allowed flow rates of more than 50 µl within 1 minute were discarded as damaged. After this initial test, PBS was pressurized by pushing a sealed column of air against the PBS at a set pressure. Fluid that flowed across dermis and into the sampling chamber was collected in 30 minute intervals by pipetting, and its mass measured. The volume of fluid was determined by converting the mass to volume via the density of PBS (1.01 g / ml).

[0161] For experiments measuring fluid flow through dermis with different water contents, dermis samples were dehydrated, fluid flow through them measured, and then dermis samples were rehydrated while mounted on a stage, after which flow across them was remeasured. To dehydrate the samples, each dermis sample of 30-mm diameter was placed on a paper towel (Essity Professional Hygiene, Philadelphia, PA) dermis-side down, and a mass of 8.9 kg was placed on top of the dermis (epidermis-side up) to drive fluid out of the dermis by convection. 25 53430309.117625-0344 GTRC 2024-159 The dermis samples were weighed before and after water removal, and the mass differential was calculated. The samples were then mounted on the apparatus, and fluid flow through the dermis measured as described above.

[0162] To measure hydraulic conductivity of the dermis samples in the hydrated state, the sample was left mounted on the apparatus, but pressure was removed, and 0.5 ml of PBS was added to the fluid sampling chamber of each sample. The apparatus was stored at 4 ºC by placing in a cold room for 20 hours, with fluid in the sampling chamber monitored and replenished as needed. The sample was then removed from the cold room, excess fluid was pipetted out of the sampling chamber, the exposed dermis area was gently padded dried with a Kimwipe, and the sample was once again subjected to pressurized PBS to push fluid through the dermis. Fluid flow through the dermis was measured as described above. Example 8: Prediction of Hydraulic Conductivity Based on a Fiber-Matrix Model

[0163] The hydraulic conductivity of dermis was modeled by considering dermis as a fiber matrix based on a model previously developed for scleral tissue in the eye with similar structure and composition. Similar to sclera, dermis is made primarily of a matrix of collagen, proteoglycans, and glycosaminoglycans containing pores through which fluid flow. Skin properties used for the model are listed in Tables 1 and 2 below.

[0164] Net hydraulic conductivity of dermis (Kdermis) was calculated by modeling the dermis as a composite medium in which two distinct domains for fluid flow are available (FIG.2). The first domain (the microscale) consisted of pores between the randomly oriented proteoglycan and glycosaminoglycan fibers in the dermis, i.e., the “ground substance”, and the hydraulic conductivity for this domain gave the hydraulic conductivity for the ground substance, Kgs. Kgswas calculated by first calculating the resistance of a medium made only of proteoglycans, then for a medium made of only glycosaminoglycans, and then adjusting the contribution of each type of fiber by the volume fraction it occupies.

[0165] The second domain (the mesoscale) consisted of a composite domain in which collagen fibers were assumed to be periodically arranged and the pores created by collagen fibers are filled with ground substance. These “lamella” had hydraulic conductivity of Klam. Finally, the net hydraulic conductivity was calculated by computing the hydraulic conductivity of a composite medium made of domains with Kgsand Klam.

[0166] Using this approach, the conductivity of the dermis, Kdermis, was calculated by solving the following equation: ^ ି^ ^^^^^^ ∙ ^^217625-0344 GTRC 2024-159 where C1 = 0.075422, C2 = 1.060283, and C3 = 0.000076, Фc is the volume fraction occupied by collagen (i.e., 17.9, calculated), and T is defined as: 1^ ^^^^ ൌ ^^^ / ^^^^1 െ ^^,^^^^^^^^^^^^^^^^ 3 ^^^ / ^^^^

[0167] An initial water though reports suggest water content in the dermis can varythe mass of the dermis. For the calculations, skin was assumed to have properties determined from literature.

[0168] Reports on the mass fraction of collagen in the dermis vary. Some works report that collagen makes up around 63% of the dry weight of the dermis, and others provide dermal fiber compositions that result in a dermal collagen content of approximately 75% of the dry weight of the dermis. For a dermis sample with 70% water mass, said dry weights result in a total collagen mass fraction (i.e., mass fraction of wet dermis) of 19% and 22.5%, respectively, which is close to the collagen content for full skin. It is worth noting that Levick reports that the total collagen mass fraction in the interstitium could be as high as 40%, which suggests a dermal water content of around 50%.

[0169] For initial calculations, a collagen composition of 67% of the dry weight of skin was assumed (i.e., 22.5% of the total mass of skin). This value was calculated by keeping the ratio of collagen to other dermal fiber in the dermis constant and normalizing by the water content. Elastin to the model was also added to the model, calculated as 10% the total mass of collagen. The mass of GAGs was assumed to be 3.5% of the mass of wet dermis.

[0170] While Levick reports that PGPs (i.e., GAG-associated proteins) can make up 8.5% of the weight of the dermis, other reports suggest that water, collagen, and elastin by themselves make up approximately 95% of the mass of lipid-free dermis. Therefore, instead of assuming that 8.5% of the mass of the dermis was PGPs, 8.5% was assumed to be non-collagenous proteins, which included PGPs, elastin, and free proteins. Given the structural and compositional similarities between the sclera and the dermis, we assumed that the mass ratio of PGPs to GAGs was 1.5:1, as done previously in a model for the sclera. Non-collagenous proteins that were not PGPs or elastin were labeled as free proteins.

[0171] Based on these assumptions, the composition for a dermal tissue sample with a water content of 70% was estimated. Next, using a 100 g mass basis and the specific volume of each component, the volume fraction occupied by each component was calculated. 27 53430309.117625-0344 GTRC 2024-159 Table 1. Mass and volume fractions of different components of the dermis at 70% hydration. issue Mass Specific Volume for Volume T fraction Volume 100 g of Fraction as avolume of 93.7 cm3, or in other words, that the density of the dermis, which is the reciprocal of the specific volume, is 1.07 g / cm3.

[0173] The contribution to hydraulic conductivity at the microscale domain was calculated (i.e., the hydraulic conductivity of the ground substance, KGS) as the hydraulic conductivity of a porous medium with two types of fibers, all randomly oriented, using the following equations. ^^ீ^^^ ି^ ^^^^ ൌ ൬ ீ^^ீ^^ ^^ ^^, ^^^^^^^^^^^^^^^^ 3where n is the number fraction (Table 1) and iis fiber radius (Table 2). The hydraulic conductivity of each fiber medium (i.e., KGAG and KPGP) was calculated using the equation below: 3^^ ^^^ൌ^^^^ ^ ^ ∙ ^െ ln^^^ீ^ீ ^ ^^^ீ^^ െ 0.931^, ^^^^^^^^^^^^^^^^ 5

[0174] below: Table 2. Radius of GAGs and PGPs in the dermis. Term Fiber type Radius (nm) rGAGGlycosaminoglycans 0.55rPGPglycans (i.e., GAG-associated proteins) 1

[0175] To calculate the contribution to hydraulic conductivity at the mesoscale, KLam, the equation below for computing the hydraulic conductivity of a medium composed of periodically 28 53430309.117625-0344 GTRC 2024-159 arranged cylinders was used. Assuming the collagen fibers are impermeable to flow, the hydraulic conductivity can be calculated as follows: ^^ଶି^ ^^^^^^ ∙ ^^^^ଷ^^ ^^^^^ ൌ ^^^^ ∙ ^1 െ 2^^^ ൭^^ ^ ^^^ െ ^^^ଶ െ ^ ^^^ൡ ^^, ^^^^^^^^^^^^^^^^ 6the. 1, as the collagen fibrils are impermeable.

[0176] To calculate the contribution to hydraulic conductivity at the macroscale, Kdermis, a variant of Equation 6 was used. This variant assumes that the collagen lamellas are only partially permeable (i.e., fluid can flow through the ground substance in the collagen lamella, but not through the collagen fibrils). This modification results in a redefinition of T as: 1^ ^^ / ^^ ൌ ^^^ ^^^^1 െ ^^ / ^ , ^^^^^^^^^^^^^^^^ 7^^^ ^^^

[0177] Therefore, the equation to calculate the composite hydraulic conductivity for the dermis as a whole (i.e., the macroscale) is as follows: ^^ଶି^ ^^ ^^ ∙ ^^^^ ^ ^^^ ^^ ^ ^^ ^ ^^^ ^^ ^^ ∙ 1 2 ଷ ^^ ^ ^^^, ^^^^^^^^^^^^^^^^ 8

[0178] After calculating the hydraulic conductivity for the dermis with a water content of 70%, the calculation was repeated for water contents between 50% and 75% in increments of 5%.

[0179] Using the hydraulic conductivity modeled as described above, the volume of ISF predicted to flow through the dermis and through pores created by microneedles to the surface of the skin was calculated.

[0180] The volumetric flow rate of ISF flowing through the dermis was calculated using Darcy’s law in one direction: ^^^ ൌ ^^ ∙ ^^^^^^ ^^ ^^^^, ^^^^^^^^^^^^^^^^ 9

[0181] The flow area was determined using the dimensions of the microneedles and by assuming the microneedles fully penetrated the skin. Therefore, each of the 10 microneedle pores had a depth of 750 µm (the height of the microneedles), a width of 200 µm, and a thickness of 50 µm. A rectangular pore shape and neglected flow through the bottom of the pores was assumed, which resulted in a total area of 3.75 ∙10-7m2for 10 pores. In all calculations, we used the viscosity of water at 37 °C (6.9∙10-4Pa∙s), a pressure gradient of -5-i0 29 53430309.117625-0344 GTRC 2024-159 kPa, and a path length of 0.13 mm. Then, the total volume flowing over a 5-minute interval was calculated by multiplying the volume flow rate (Q̇) by the time interval, Δt. Example 9: Thermoporometry

[0182] Pore size distribution in skin samples was estimated from thermograms collected by differential scanning calorimetry (DSC) using thermoporometry, a technique based on the melting point depression of water confined in small pores. This method has been previously used to measure pore sizes in hydrogels.

[0183] Four-millimeter biopsies of pig ear skin with original mass of 14-20 mg were submerged in 25 ml of PBS at 4 °C for 20 hours. The samples were then removed, gently blotted dry, and their mass recorded again. Next, each sample was placed in an aluminum hermetic DSC pan, sealed, and placed in a TA DSC 250 (TA Instruments, New Castle, DE).

[0184] The DSC scanning program consisted of an equilibration step to bring the sample to - 30 °C, a 5-minute isothermal step at -30 °C, and a ramp at 0.5 °C / minute from -30 °C to 5 °C. After the scan, the skin sample was removed from the pan and partially dehydrated by pressing against a Tork paper towel (Essity) until 2-3 mg of mass was removed from the sample. The weight after dehydration was recorded, and the sample was put again in a sealed DSC pan and rescanned. This process was repeated up to four times, after which the sample was fully dehydrated by lyophilization and scanned in the DSC again. There was little variation in the curves due to freeze-thaw cycles. For all samples, peak onset of melting and enthalpy of melting were calculated using TRIOS software (TA instruments), and the thermogram data exported to Excel (Microsoft, Redmond, WA) to calculate the amount of water in pores of different sizes. Skin samples with lower water content showed smaller peaks of heat flow (i.e., enthalpy change), which is consistent with the decreased amount of water in the skin. Lyophilized skin showed no peak, which supports the interpretation that the peaks in the thermograms correspond to the melting of water in the samples.

[0185] To calculate the fraction of freezable water in skin samples, the total enthalpy calculated from the TRIOS software for each sample was divided by the specific melting enthalpy of water (334 J / g). The difference between the freezable water in each sample and the total water in each sample (known from lyophilization) was deemed non-freezable water.

[0186] To calculate the amount of water in pores of different sizes (i.e., the size distribution of water-filled pores in the skin), the cumulative enthalpy of the water melting peak in the sample was first calculated by integrating the heat flow vs. temperature curve. Next, the temperatures in the DSC scan were converted to pore sizes, but using an empirical offset of -1.1 °C that corrected for peak onset of water melting in the equipment used for the experiments. 30 53430309.117625-0344 GTRC 2024-159 Then, the enthalpy of water in pores in a particular size range was determined by subtracting the cumulative enthalpy for the smaller pore (which corresponds to a lower temperature and therefore a lower cumulative enthalpy) from the cumulative enthalpy for the larger pore (which corresponds to a higher temperature and therefore a higher cumulative enthalpy). This enthalpy difference was then divided by the total cumulative enthalpy, which gave the fraction of freezable water in pores within said size range. Finally, multiplying this fraction by the total mass of freezable water in the sample gave the net amount of water within pores in the size range. Example 10: ISF Extraction from Pig Skin by Pressure-Driven Flow

[0187] The effect of positive or negative pressure applied to skin during ISF extraction after skin puncture by microneedles was assessed (FIGS.7A-7B). After microneedle puncture but without applied pressure, only 0.003 ± 0.005 µl / microneedle were extracted from the skin. Applying suction increased ISF extraction in a roughly linear fashion, with a 5-fold increase in ISF extracted due to a 7.5-fold increase in suction pressure from -10 kPa to -75 kPa (p<0.004) (FIG.7C). In contrast, applying positive pressure up to 400 kPa had no significant effect on ISF extracted ( p>0.97); a pressure of 800 kPa was needed to significantly increase ISF extracted (p<0.009) (FIG.7D). While both positive and negative pressure provide a driving force for ISF flow, positive pressure may compress tissue, while suction may expand tissue, which could explain the greater efficacy of suction for ISF extraction. Example 11: Effect of Physical Parameters on ISF Extraction from Pig Skin

[0188] The effect of different physical parameters on the volume of ISF extracted via pressure-driven flow was assessed. Parameters that could potentially be controlled in a clinical ISF extraction system by design features of the system or by selection of the treatment site were chosen. The effects of the presence of microneedle pores, microneedle length, suction source, and skin stretching, temperature, and thickness were assessed.

[0189] It was determined that microneedle-created pores are important for extracting ISF, as shown by an 11-fold increase in ISF extracted from skin punctured with microneedles versus intact skin (p=0.04) (FIG.8A). It was also found that the depth of microneedle pores did not significantly affect volume of ISF extracted by comparing skin puncture using microneedles measuring 250 µm versus 750 µm long (p=0.63) (FIG.8B). This suggests that puncturing epidermis (~50-100 µm thick) was sufficient to extract ISF from the skin, and deeper penetration into dermis was not needed.

[0190] Instead of the bulky laboratory pump used for most of our studies, it was found that a small, hand-held cosmetic pump operated at approximately the same suction pressure yielded 31 53430309.117625-0344 GTRC 2024-159 similar amounts of ISF extracted (p=0.44) (FIG.8C). This finding may enable use of a small, low-cost and convenient device to facilitate widespread use.

[0191] In the absence of effective pressure (≤200 kPa), it was found that skin stretching had no effect on ISF extraction (p>0.06), but at high pressure (800 kPa), stretching helped (p<0.0001) (FIG.8D). Increased skin tension due to stretching could open skin pores and more- effectively spread the applied pressure gradient across skin to drive ISF flow.

[0192] There was a small but significant increase in ISF extracted at 37 °C versus room temperature (p=0.017) (FIG.2E), suggesting a role of temperature-induced changes in ISF viscosity, skin hydraulic conductivity or other properties. Finally, volume of ISF extracted was not correlated to skin thickness (R2<0.10) (FIG.2F), which indicates that the greater ISF content of thicker skin did not facilitate ISF extraction. Example 12: Effect of Skin Water Content on ISF Extraction from Pig Skin

[0193] It was hypothesized that increased skin water content could increase ISF extraction by two possible mechanisms. First, increased absolute water content means the presence of more ISF that can be extracted. However, the total amount of ISF extracted in these examples (<10 µL / cm2) was only a small fraction of ISF in skin (70 µL / cm2for skin of 1mm thickness and 70% hydration), which suggests that total ISF content may not be limiting.

[0194] The second mechanism is based on ISF extraction being limited by resistance to flow through the porous medium of dermis, which is comprised of collagen and elastin fibers surrounded by an extracellular matrix of proteoglycan fibers. Increased percent water content means larger pores that have less resistance to flow and possibly a larger number of pores that provide more pathways for flow.

[0195] To test this hypothesis, skin water content was varied from 67.0 ± 5.0% to 57.7 ± 4.3% by dehydrating skin for up to 20 minutes (p<0.0001) (FIG.9A) and it was found that average ISF volume extracted decreased 7-fold (p<0.0001) (FIG.9B). Examining all 62 individual data points with skin water content ranging from 51% to 74% and ISF volume extracted varying from 0.01 to 4.9 µl, it was similarly found that ISF volume increased with increasing skin water content (p<0.01) (FIG.9C). Skin having <60% water content generally produced <0.1 µl of ISF, and skin with >65% water content produced volumes as high as 4.9 µl.

[0196] Guided by the hypothesis that ISF extraction is limited by flow through dermis, the skin was modeled as a porous fiber matrix; the model had no fitted parameters and its structure was based on skin’s fiber content and structure. The modeled hydraulic conductivity of dermis (FIG.9C) increased with increasing skin water content due primarily to expansion of water- filled pores in dermis and exhibited a similar functionality to the empirical fit of ISF extracted. 32 53430309.117625-0344 GTRC 2024-159 The hydraulic conductivity model was extended to predict ISF volume extracted through microneedle pores, which similarly increased with increasing skin water content, although less steeply than the empirical data fit (FIG.9C). Altogether, these data show that increased skin water content increased ISF extraction by a mechanism that is hypothesized to involve increased pore size in dermis. Example 13: Effect of Enzyme Treatment on ISF Extracted from Pig Skin

[0197] Based on the expectation that ISF extraction is limited by flow through a porous matrix of dermal fibers, it was found that degradation of skin’s most-abundant fibers using collagenase and hyaluronidase was able to increase ISF extraction in a concentration-dependent manner by as much as 6-fold (p<0.0001) (FIGS.10A, 11). Combination of the enzymes did not further increase the magnitude of the effect (FIG.10B). As a negative control, heat-denatured collagenase did not increase the volume of ISF extracted (FIG.10C). Other types of collagenases similarly increased ISF volume extracted (FIG.10D). Altogether, these data indicate that collagen and hyaluronic acid provide significant barriers to ISF flow, and that enzymes can break down those barriers.

[0198] The fiber matrix model was used to predict the effect of changing the collagen and hyaluronic acid (i.e., GAG) fibers in the dermis on dermal hydraulic conductivity (FIG.11). The model predicted a larger increase in hydraulic conductivity from a decrease in GAG concentration than from a decrease in collagen concentration. Therefore, a larger increase in ISF collected from treating the skin with hyaluronidase was expected than from treating the skin with collagenase. The discrepancy could be explained by the fact that the enzyme treatment protocol did not necessarily change the concentration of fibers in the skin, but only degraded sections of the fiber matrix. This fiber degradation method likely produces new pores without changing the total concentration of fibers of each type, and therefore it is possible that the mechanism for the ISF increase experimentally produced by collagenase or hyaluronidase treatments were not within the scope of the model. Example 14: Effect of Pressure and Dermal Water Content on Hydraulic Conductivity of Dermis

[0199] Because ISF extraction depends on hydraulic conductivity of dermis, the effect of pressure and skin water content on skin’s hydraulic conductivity was directly measured. As a first assessment, the hydraulic conductivity of dermis was compared to that of full skin that included intact epidermis with stratum corneum (FIG.12A). As expected, hydraulic conductivity of full skin was almost 20-fold lower than that of dermis alone (p=0.001), 33 53430309.117625-0344 GTRC 2024-159 consistent with observations that piercing epidermis with microneedles dramatically increases ISF extraction from skin.

[0200] Pressure also increased effective dermal hydraulic conductivity, exhibiting >3-fold increase from 34 to 200 kPa (p<0.0001) (FIGS.12B, 5-6), suggesting possible alteration of pore structures in dermis by applied pressure. Extrapolation of the hydraulic conductivity curve to zero pressure yielded a conductivity of 7x10-16m2 / (Pa∙s), in good agreement with prior literature. Reducing dermal water content (FIGS.12C, 13A) also decreased dermal hydraulic conductivity (p<0.0001) (FIGS.12D, 13B), which is consistent with observed effects of water content on ISF extraction (FIG.9). Altogether, this suggests that pressure-driven ISF extraction from skin is a complex function of driving force, dermal water content, and skin barrier modification.

[0201] It was found that decreasing dermal water content by ≥40% (FIGS.12C, 13A) resulted in an effective dermal hydraulic conductivity 50% lower than in hydrated samples (p<0.05) (FIGS.12D, 13B). In FIG.13A, “before dehydration” refers to the mass of the dermis after epidermis removal and prior to any hydraulic conductivity measurement. “After dehydration” indicates the mass of the dermis after the dehydrating procedure, which was also prior to any hydraulic conductivity measurements. The thickness of these samples was 0.63 ± 0.13 mm (N=10).

[0202] For measurement of the hydraulic conductivity of hydrated dermis (FIG.13B), the thickness of each dermis sample at the time of the measurement (i.e., after rehydration) was assumed to be equal to the thickness of dermis before dehydration (i.e., the mass of each bar in FIG.13A was used to calculate the thickness of each sample in the “hydrated” state). This assumption was validated by comparing the original thickness of dermis samples (0.94 ± 0.15 mm, N=10) to the thickness of 8-mm dermis biopsies taken at the end of the experiment (i.e., after rehydration; 1.10 ± 0.19 mm, N=7) (p>0.05). Both of these groups were different to the dehydrated sample (p<0.0001 for both).

[0203] Prior data (FIG.9C) showed that a similar decrease in skin water content resulted in a 7-fold drop in volume of ISF collected (FIG.9C), which is a larger fold-drop than the drop hydraulic conductivity. A potential explanation is that the process of collecting fluid during measurements of hydraulic conductivity required the flow of water through the dermis for extended periods of time, which might rehydrate the dermis during the process.

[0204] Thermoporometry was used to estimate the distribution of water-filled pores in the skin. In thermoporometry, the temperature depression of the melting point of frozen water confined in small pores can be used to calculate the amount of water in pores of different sizes. 34 53430309.117625-0344 GTRC 2024-159 The melting point depression, ΔT, experienced by water in a pore with radius rp can be estimated using the following equation: 2T^^^ ^^^^^^^^ ΔT ൌ ^^∙ ^^Δ^^, ^^^^^^^^^^^^^^^^ 10^ ^^^^

[0205] The values used in the table below. Table 3. Valuesof water in pores of different sizes in Equation 10. Property Description Value ΔT (K) Temperature depression of the melting point of water be calculated T0(K) Melting point of unconfined solid273.15γls(N / m) Liquid-solid surface tension 0.039 (estimated) ρl(kg / m3) Density of liquid 1.0 ΔHf(J / kg) Molar heat of fusion -334 θ (°) Contact angle between liquid and solid 0*rp(m) Radius of pore Various*assumed for pores with water boundary layer

[0206] Using Equation 10 to calculate the temperature depression corresponding to pore diameter between 14 nm and 1000 nm produced the curve shown in FIG.14. Example 15: Effect of Skin Water Content on Skin Pore Size

[0207] It was hypothesized that reduced ISF extraction and dermal hydraulic conductivity due to decreased skin water content can be explained by a reduction of size of dermal pores that provides greater resistance to flow. To test this hypothesis, pore size distribution in skin samples was determined with different water content using thermoporometry, which is a technique that determines pore size based on melting point depression of water confined in small pores.

[0208] Differential scanning calorimetry (DSC) analysis (FIG.15A) showed that enthalpy change of water increased monotonically from ~80 J / g to ~200 J / g for skin with 45% to 74% water content (p<0.0001) (FIGS.15B-15C). Measured enthalpy changes were used to calculate mass of freezable water in skin, which increased from ~30% to ~60%, and mass of non- freezable water, which remained relatively constant between 15% and 20% (p=0.11) (FIG. 15C). If non-freezable water is interpreted as bound to the edges of pores, the fact that the amount of non-freezable water remained relatively constant may mean that total number of pores likewise did not change. If freezable water is interpreted as being in the center of pores and able to flow, the increase in freezable water with increasing water content suggests growth of pore diameter with more mobile water. 35 53430309.117625-0344 GTRC 2024-159

[0209] The shifts in thermogram peaks to higher temperatures as skin water content increased (FIGS.15A-15B) corresponds to a shift to larger pore size. Using these data, it was found that as skin water content increased, water content in the largest pores (>1000 nm) also increased (p<0.0001), while water content in smaller pores did not change significantly (p>0.05) (FIG.15D). Median pore size in skin increased ~5-fold, going from 63 ± 7 nm at the lowest water content to 320 ± 109 nm at the highest (FIG.15E). These data further support the hypothesis that increased skin water content increases skin pore size, which then increases dermal hydraulic conductivity and ISF extraction from skin.

[0210] To determine if there was non-negligible freezable water freezing below -30 °C, skin samples from -60 °C to 5 °C were scanned (FIG.16A). The resulting thermograms show that the DSC thermograms were flat from -60 °C to approximately -10°C, indicating negligible amounts of water in pores with diameters 15 nm or less.

[0211] To determine if repeated freeze-thaw cycles might change the thermograms seen during thermoporometry measurements and thereby create an experimental artifact, three skin samples were scanned four times in the DSC, thereby subjecting the samples to four freeze-thaw cycles. The resulting thermograms, cumulative enthalpies, and pore size distribution (FIGS. 16B-16D) show that there was negligible variation in the curves and distributions after each freeze-thaw thaw cycle. Example 16: Human Studies

[0212] All procedures were approved by the Georgia Institute of Technology Institutional Review Board, and written informed consent was obtained from all subjects before the study. A total of 23 healthy study subjects were recruited from among students, staff, or alumni of the Georgia Institute of Technology with demographics shown in Table 4. Participation was open to any healthy adult. Exclusion criteria included serious dermatological conditions, pregnancy, immunodeficiency, or recent blood donation. Table 4. Demographics of 23 participants involved in laser-based ISF extraction studies. Age (years) M D 2753430309.117625-0344 GTRC 2024-159 BMI (kg / m2)*Mean (SD) 21.6 (3.3) M di I R 216 207 – 239

[0213] ect’s forearm skin was cleaned with an alcohol swab, and a Tegaderm film (3M, Saint Paul, MN) with three 12 mm diameter holes cut in it was applied to delineate three treatment sites for sequential and identical application of laser, microneedle array, and suction; each group of three sites is referred to as a “treatment area”. Up to four treatment areas were used per subject. Control Gauze

[0214] Prior to laser or microneedle array application, the surface of the three skin sites was swabbed for ~30 seconds with a sterile medical gauze (Ultrapure nonwoven sponges, Crosstex International, Hauppauge, NY) wetted with 5-10 µl of sterile water. This sample was used to determine background biomarker levels on the skin surface. The sample was stored in a LoBind Eppendorf tube (Eppendorf, Hamburg, Germany) and frozen at -20 °C until further use. Laser Application

[0215] A Palovia Skin Renewing fractional laser (Palomar Medical Technologies, Burlington, MA) was used to pretreat the skin. This cosmetic laser uses 10 ms micropulses of 1410 nm light that generate micro-sterile inflammation in small areas of the skin.

[0216] To apply the laser on human subjects, the laser power level was set to “high”, aligned with a skin site delineated by the Tegaderm, and pressed to emit a “pulse” comprised of a series of 54 laser micropulses, each micropulse with energy of 2.0 ± 0.2 mJ (FIGS.17-18). Each pulse treated an area with diameter of ~200 µm, and all 54 pulses fit within a total skin area of ~55 mm2. Depending on the treatment selected for a particular treatment area, either zero, one, four, or eight laser pulses were applied to all three sites. Unless otherwise noted, there was a delay of 5 minutes between initiation of laser pulses and initiation of suction. The eight-pulse treatment (24 pulses total among three skin sites) took approximately 3 minutes, whereas the single-pulse treatment took approximately 15 seconds (for all three skin sites).

[0217] The power output of the Palovia fractional laser was measured using an Ophir Vega laser power meter (Ophir Optronics, Jerusalem, Israel) connected to a PE25-C general purpose pyroelectric sensor (Ophir Optronics). Each laser pulse consisted of a set of sequential micropulses emitted by the laser upon pressing the activation button. As shown in FIG.17, each micropulses had an energy roughly ranging between 1.5 and 2.0 mJ.

[0218] The energy outputs at low, medium, and high settings of the laser were 1.9 ± 0.2, 2.0 ± 0.1, and 2.0 ± 0.2 mJ, respectively (FIG.18). While the energy of each micropulse remained 37 53430309.117625-0344 GTRC 2024-159 similar at each energy setting, the number of micropulses per pulse at the low, medium, and high energy settings were 35, 40, and 54, respectively. Microneedle Array Application

[0219] After applying the laser pulses, a five-microneedle array with needles 250 µm long was applied 20 times to each skin site to create 100 micropores or 40 times to create 200 micropores in each of the three sites. For all treatment areas in which laser pulse(s) were applied, suction was applied 5 minutes after the first laser pulse. For treatment areas in which no laser pulse was applied, suction was applied 5 minutes after microneedle insertion. Vacuum Application and ISF Extraction

[0220] Vacuum was applied using a Negative Pressure Cutaneous Suction system (NP-2, Electronic Diversities, Finksburg, MD). The vacuum cup was placed over the skin sites treated with the laser and the microneedle array. The vacuum cup had three 8-mm-diameter orifices that were aligned with the three holes in the Tegaderm at the application site. Vacuum was applied to the skin by slowly ramping the suction from 0 to -50 kPa over ~3 minutes. The suction was stopped after 5 minutes and the ISF on the skin surface was collected by rubbing the skin surface with a piece of sterile medical gauze (Crosstex International) wetted with 5-10 µl of sterile water for 30 seconds.

[0221] In some cases, this process was repeated at the same skin location once more after another 5 minutes of suction, after which ISF was extracted, and then after another 10 minutes of suction, after which ISF was extracted, to enable three ISF extractions over a cumulative suction time of 20 minutes. In some cases, continuous suction was applied for 20 minutes and only extracted ISF at the end. For the 20-minute ISF extraction studies, the procedure time from laser application to end of suction and ISF extraction for all treated areas was up to 35 minutes. In some cases, subjects returned for visual examination and imaging of the treated skin sites for up to 4 weeks. Pain Scores

[0222] Pain scores for the laser treatment, microneedle insertion, and suction treatments were determined using a visual analogue scale (VAS) immediately after each procedure was performed. In one study, pain scores were normalized to the insertion of a 21G needle (Becton Dickinson, Franklin Lakes, NJ) into the forearm to a depth of 5 mm, which was performed at a random time point during the study. In another study, pain scores were normalized to a lancet puncture into the skin of a fingertip. 38 53430309.117625-0344 GTRC 2024-159 Capillary Blood Collection

[0223] In some cases, capillary blood from the subject fingers were collected by lancet puncture (CVS Health, Woonsocket, RI) on the fingertip of the middle or ring finger during the beginning of the 10-minute suction interval of the 20-minute ISF extraction protocol. Approximately 50-100 µl of blood were collected from each subject in BD Microtainer Capillary Blood Collectors with Gold Color Closure (Becton Dickinson). Optical Coherence Tomography (OCT) Imaging

[0224] OCT imaging was used to study the effect of the laser on skin. OCT images of the skin were taken using a VivoSight Dx (Michelson Diagnostics, Kent, UK) that used infrared light (1305 nm) to visualize skin at depths up to 500 µm from the skin surface. OCT scan settings were set to produce 250 slices of 2 µm thickness in a 6 mm x 6 mm section of the skin. When assessing blood flow and epidermal thickness analysis, the OCT was set to “dynamic scan”. The companion VivoTools software (Michelson Diagnostics) was used to estimate blood vessel plexus depth and epidermal thickness in some subjects. Measuring the Power of the Cosmetic Fractional Laser

[0225] The power output of the Palovia fractional laser was measured using an Ophir Vega laser power meter (Ophir Optronics, Jerusalem, Israel) connected to a PE25-C general purpose pyroelectric sensor (Ophir Optronics). A custom-made adapter was added on top of the sensor to position the laser, and the laser was pressed against the adapter. The path between the laser active area and the sensor active area was air. Example 17: Analytical Techniques Blood Processing and Serum Separation

[0226] The volume of blood collected during fingerstick sampling of capillary blood was estimated visually from the markings of the vial. Serum was separated from blood by spinning the tubes at 16.1 g for 10 minutes in an Eppendorf centrifuge 5415 RH, and then collected in 0.5 ml Eppendorf Protein LoBind tubes. Determination of ISF Volume

[0227] ISF volume was determined by measuring the concentration of sodium in the fluid. The sodium concentration was determined electrochemically using an ion selective electrode (PerfectION comb Na+, Mettler Toledo, Columbus, OH). The concentration of sodium in ISF was assumed to have a constant value in health individuals of 140 mM, enabling determination of ISF volume as the product of the measured sodium concentration in fluid swabbed from the skin surface and the volume of fluid swabbed from the skin surface (5-10 µl) divided by the concentration of sodium in ISF (140 mM). The volume measured from the ISF sample was 39 53430309.117625-0344 GTRC 2024-159 corrected by subtracting the background sodium on the skin surface, which was measured from a skin surface swab prior to application of microneedle and suction (see above). Determination of Plasma and ISF Protein Concentration

[0228] Protein concentration was determined using a MicroBCA assay (ThermoFisher Scientific) to measure the total protein content of plasma and ISF samples. After the total protein content for each sample was measured, it was divided by the total sample volume of plasma or ISF (as determined using the sodium assay) to obtain the concentration of protein in the plasma or ISF extracted). Example 18: Recruiting ISF in Human Subjects by Increasing Skin Water Content

[0229] Guided by the hypothesis that increased skin water content can facilitate ISF extraction, a skin pretreatment to locally increase skin water content for safe use in humans was designed. The pretreatment involved minor skin injury by an over-the-counter fractional cosmetic laser used to reduce the appearance of wrinkles (FIGS.20-21). When administered to human subjects, each laser pulse generated 54 microthermal zones of <0.5 mm diameter within a skin area of 55 mm2that produced localized edema that was visible to the naked eye within 3 minutes, peaked at ~5 minutes, and mostly subsided within 20 minutes, leaving only mild erythema that disappeared within a day. Application of 8 laser pulses produced larger and more prolonged edema.

[0230] Further characterization by optical coherence tomography (OCT) showed distinct spots produced by the laser on the skin surface of the human subjects (FIG.19, FIG.20), and optical cross-sectioning through these spots showed dark regions in epidermis and superficial dermis, interpreted as fluid-rich regions created by the laser that appear dark due to lack of light scattering, as opposed to cell- and fiber-rich regions that scatter light and therefore appear white (FIG.19).

[0231] To further confirm that laser treatment increased skin water content in vivo, we exposed rats to the same laser treatment performed on human subjects. Edema in rat skin was less pronounced than in humans and was accompanied by milder erythema. Measurement in skin biopsies demonstrated a modest, but significant increase in skin water content of 3% due to laser treatment (p<0.001) (FIGS.21A-21C), consistent with the findings in human subjects.

[0232] Application of fractional laser produced a well-defined edema in the skin of human subjects. Visualization via optical coherence tomography (OCT) also showed well-defined locations where the laser interacted with the skin (FIG.20).

[0233] However, application of the laser to rat skin did not produce edema as clearly. Explanations for the lack of visual appearance of edema in rat skin likely include the smaller 40 53430309.117625-0344 GTRC 2024-159 dermis thickness of rat skin, and the optimization of the cosmetic laser device to focus the laser beam to the appropriate layer of human skin. Despite the lack of visual appearance of edema, measurement of the skin water content of rat skin treated and not treated with laser (FIGS.21A- 21C) showed that the laser produced a net increase in water content of 3% (p<0.001). Example 19: Extracting ISF from Human Subjects After Recruiting ISF

[0234] ISF was extracted from the skin of human subjects using laser, microneedles, and suction. ISF collected with or without laser treatment appeared as a clear yellowish fluid on the skin surface. Applying 0, 1, 4, or 8 laser pulses before microneedle application and suction for 5 minutes progressively increased ISF extracted from 0.66 ± 0.81 µl to 5.3 ± 4.0 µl, which shows that laser treatment increased ISF volume by up to a factor of 8.0 (p=0.003) (FIG.22B). Similarly enhanced ISF extraction was the same if microneedles and suction were administered immediately after laser treatment or after a 5-min delay (p>0.05) (FIG.23). Subjects reported only minor discomfort with the ISF extraction process, which produced less pain than insertion of a hypodermic needle (FIG.22C).

[0235] In contrast to ISF extraction in humans, laser treatment in pig skin ex vivo provided no increase in ISF extraction (p>0.05) (FIG.22A), consistent with the hypothesis that enhanced ISF extraction was due to edema, which only occurs in vivo.

[0236] To further increase ISF volumes, ISF was extracted under suction for up to 20 minutes in another cohort of human subjects. It was again found that ISF volume increased with number of laser pulses (ANOVA, p=0.018), and also increased with duration of suction (p=0.003), ranging from 11 ± 12 µl of ISF after 5 min to 55 ± 49 µl after 20 minute suction coupled with 8 laser pulses, which is a 3.5-fold increase compared to no laser (p=0.013) (FIG. 22D). The largest ISF extraction from any subject was 130 µl, which is 8-fold greater than the average ISF volume extracted over 20 minutes without pulses. Applying suction for 20 minutes continuously or as three sequential suction periods yielded similar ISF volumes (p=0.55) (FIG. 22D), indicating that intermittent ISF sampling during extended extraction did not limit ISF extraction. OCT imaging after the ISF extraction process showed fluid-rich regions ~100-200 µm deep in epidermis and superficial dermis (FIG.24). Subjects again reported only minor discomfort with ISF extraction, which produced pain comparable to that of a lancet puncture (FIG.22E).

[0237] Total protein concentration in ISF was ~60% of that in plasma (p<≤0.05 for all pairs) (FIG.22F), which is similar to prior studies showing reduced protein concentration in ISF, and the concentration did not depend on suction time (p>0.05 for all groups) or number of laser 41 53430309.117625-0344 GTRC 2024-159 pulses (p>0.05) (FIG.22G, FIG.25A-25B). This suggests that ISF collected after laser treatment may have protein content similar to native ISF.

[0238] Overall, ISF extraction was well tolerated. While edema was not visible after ISF extraction without laser, mild, highly localized edema was present after laser treatment, which subsided in ~1 hour. Mild erythema was seen after ISF extraction with or without laser treatment. The edge of the suction device left a ring-shaped mark on skin, which generally disappeared within hours, but sometimes persisted for up to 7 days. While ISF extraction mostly produced clear fluid, sometimes small drops (~1 µl) of blood appeared, apparently from individual micropores. A faint grid pattern of laser spots and skin hyperpigmentation were seen immediately after treatment and generally disappeared within a day after one laser pulse, but persisted ≤1 week after 4 pulses and ≤4 weeks after 8 pulses for the subjects treated with suction. No other adverse events were noted. Embodiments

[0239] Some embodiments of the present disclosure are described in view of one or more of the following:

[0240] Embodiment 1. A method comprising: applying energy into a selected region of a subject’s skin in an amount effective to facilitate interstitial fluid (ISF) mobilization to and / or within the selected region; inserting an array of microneedles into the subject’s skin at an insertion site to form a plurality of micropores in the subject’s skin in the selected region; and collecting ISF from the insertion site.

[0241] Embodiment 2. The method of Embodiment 1, wherein the collecting ISF comprises collecting ISF that has flowed from the micropores onto the surface of the subject’s skin or into a fluidic system or reservoir positioned on the subject’s skin at the insertion site, optionally wherein the collected ISF contacts a sensor that makes a measurement of the composition or other property of the ISF.

[0242] Embodiment 3. The method of Embodiment 1 or 2, wherein the collecting ISF comprises collecting ISF via the microneedles which remain inserted at the insertion site.

[0243] Embodiment 4. The method of any one of Embodiments 1 to 3, wherein the microneedles are hollow or porous.

[0244] Embodiment 5. The method of any one of Embodiments 1 to 4, wherein the microneedles comprise a hydrogel material, which is configured to swell with collected ISF.

[0245] Embodiment 6. The method of any one of Embodiments 1 to 5, wherein the array of microneedles is configured to penetrate at least across the stratum corneum and the viable epidermis, and into the dermis. 42 53430309.117625-0344 GTRC 2024-159

[0246] Embodiment 7. The method of Embodiment 6, wherein the array of microneedles is configured to penetrate into the papillary dermis.

[0247] Embodiment 8. The method of Embodiment 6, wherein the array of microneedles is configured to penetrate into the reticular dermis.

[0248] Embodiment 9. The method of any one of Embodiments 1 to 8, wherein the microneedles have a length from about 100 µm to about 2 mm, such as from 250 µm to 750 µm.

[0249] Embodiment 10. The method of any one of Embodiments 1 to 9, wherein the array of microneedles comprises from 2 microneedles to 1000 microneedles, such as from 5 to 500 microneedles.

[0250] Embodiment 11. The method of any one of Embodiments 1 to 10, wherein the array of microneedles is inserted into the subject’s skin, and then one or more times removed from the subject’s skin and reinserted into the subject’s skin in the selected region.

[0251] Embodiment 12. The method of Embodiment 11, wherein sequential insertions of the array of microneedles occur within an interval ranging from 1 second to 1 minute to create at least twice as many micropores as the number of microneedles in the array.

[0252] Embodiment 13. The method of Embodiment 11 or 12, wherein a sequential insertion of the array of microneedles occurs following an interval ranging from 10 minutes to 24 hours to create new micropores after at least some of the previously created micropores have closed and / or their ability to pass ISF therethrough has become degraded.

[0253] Embodiment 14. The method of any one of Embodiments 1 to 8, wherein the array of microneedles remains inserted in the skin for a period ranging from 1 minute to 24 hours, such as 5 minutes to 30 minutes, or 30 minutes to 6 hours.

[0254] Embodiment 15. The method of any one of Embodiments 1 to 14, wherein from 2 to 4000 micropores, such as from 20 to 500 micropores, are formed in the selected region of the subject’s skin.

[0255] Embodiment 16. The method of Embodiment 15, wherein the micropores initially are between 10 µm and 1000 µm deep, and between 10 µm and 300 µm wide, or wherein the cross-sectional area of each micropore initially ranges from about 0.01 mm2to about 0.1 mm2.

[0256] Embodiment 17. The method of any one of Embodiments 1 to 16, wherein the array of microneedles is no longer inserted in the skin at the time of the collection of ISF from the subject’s skin.

[0257] Embodiment 18. The method of any one of Embodiments 1 to 16, wherein the array of microneedles remains within the skin during the collection of ISF from the subject’s skin. 43 53430309.117625-0344 GTRC 2024-159

[0258] Embodiment 19. The method of any one of Embodiments 1 to 18, wherein the application of energy is effective to induce local edema within the selected region of the subject’s skin.

[0259] Embodiment 20. The method of one of Embodiments 1 to 19, wherein the total energy applied is in an amount effective to produce sustained edema lasting more than 15 minutes.

[0260] Embodiment 21. The method of any one of Embodiments 1 to 20, wherein the selected region is in an area of the subject’s skin which is about 0.1 cm2to 100 cm2, such as from 1 cm2to 10 cm2.

[0261] Embodiment 22. The method of any one of Embodiments 1 to 21, wherein the energy applied into the selected region is applied in one or more energy increments of from 1 microsecond to 10 minutes.

[0262] Embodiment 23. The method of any one of Embodiments 1 to 22, wherein the energy applied into the selected region is applied in two or more energy exposures, such as from 2 to 2000 energy exposures, or from 5 to 100 energy exposures.

[0263] Embodiment 24. The method of Embodiment 23, wherein the two or more energy exposures are applied over a period from 1 second to 1 day.

[0264] Embodiment 25. The method of any one of Embodiments 1 to 24, wherein the energy is applied to the selected region from an energy source selected from a light source, such as an ultraviolet light source, a visible light source, an infrared light source, or a laser.

[0265] Embodiment 26. The method of any one of Embodiments 23 to 25, wherein between each energy exposure, the energy source is removed from and repositioned on the subject's skin in the selected region.

[0266] Embodiment 27. The method of any one of Embodiments 1 to 26, wherein each energy exposure creates a fractional array of energy deposits within the selected region.

[0267] Embodiment 28. The method of Embodiment 27, wherein each of the fractional arrays of energy deposits are not directly overlapping.

[0268] Embodiment 29. The method of any one of Embodiments 23 to 25, wherein between each energy exposure, the energy source remains in place on the subject's skin in the selected region.

[0269] Embodiment 30. The method of any one of Embodiments 1 to 26, wherein each energy exposure creates a uniform array of energy deposits within the selected region.

[0270] Embodiment 31. The method of any one of Embodiments 25 to 30, wherein the energy source is a laser. 44 53430309.117625-0344 GTRC 2024-159

[0271] Embodiment 32. The method of any one of Embodiments 25 to 31, wherein the energy from the light source has a wavelength ranging from 100 nm to 2100 nm.

[0272] Embodiment 33. The method of any one of Embodiments 25 to 32, wherein the energy from the light source has a pulsing frequency ranging from 1 Hz and 30 Hz.

[0273] Embodiment 34. The method of any one of Embodiments 23 to 33, wherein each energy exposure has an energy output ranging from 0.1 J per cm2to 20 J per cm2of surface of skin in the selected region.

[0274] Embodiment 35. The method of any one of Embodiments 1 to 24, wherein the energy is applied to the selected region by radiofrequency (RF) waves, by ultrasound, and / or by electrical current.

[0275] Embodiment 36. The method of any one of Embodiments 1 to 24, wherein the energy is applied to the selected region by mechanical means.

[0276] Embodiment 37. The method of any one of Embodiments 1 to 36, wherein the application of energy is effective to generate heat within the epidermis and / or the dermis of the subject’s skin.

[0277] Embodiment 38. The method of any one of Embodiments 1 to 37, wherein the collecting ISF that flows from the micropores to the surface of the subject’s skin comprises inducing flow of ISF within the subject’s skin by the application of a pressure gradient to the subject’s skin.

[0278] Embodiment 39. The method of Embodiment 38, wherein the pressure gradient comprises application of a positive pressure in a range from 10 kPa to 1500 kPa, such as from 100 kPa to 800 kPa.

[0279] Embodiment 40. The method of Embodiment 38, wherein the pressure gradient comprises application of a negative pressure in a range from -5 kPa to -100 kPa, such as from - 30 kPa to -80 kPa.

[0280] Embodiment 41. The method of any one of Embodiments 38 to 40, wherein the pressure gradient is applied to the periphery of the selected region.

[0281] Embodiment 42. The method of any one of Embodiments 38 to 40, wherein the pressure gradient is applied to the periphery of each of the micropores in the subject’s skin in the selected region.

[0282] Embodiment 43. The method of any one of Embodiments 38 to 40, wherein the pressure gradient is applied to substantially all of the selected region. 45 53430309.117625-0344 GTRC 2024-159

[0283] Embodiment 44. The method of any one of Embodiments 38 to 43, wherein the application of a pressure gradient comprises repeated intermittent application of positive or negative pressure, such as at frequency ranging from about 0.2 Hz to about 30 Hz.

[0284] Embodiment 45. The method of any one of Embodiments 1 to 44, wherein the collecting ISF that flows from the micropores to the surface of the subject’s skin is carried out following a delay period after the steps of applying energy and the inserting the array of microneedles.

[0285] Embodiment 46. The method of Embodiment 45, wherein the delay period is in a range from 1 minute to 24 hours, such as from 2 minutes to 10 minutes.

[0286] Embodiment 47. The method of Embodiment 45 or 46, wherein the surface of the subject’s skin about the selected area is cleaned during the delay period.

[0287] Embodiment 48. The method of any one of Embodiments 1 to 47, wherein the ISF collected is in an amount from 1 µl to 100 µl, such as 10 µl to 60 µl.

[0288] Embodiment 49. The method of any one of Embodiments 1 to 48, wherein the average rate of ISF collection over a total collection period for the subject is between 0.1 µl / minute and 100 µl / minute, such as 1 µl / minute to 20 µl / minute.

[0289] Embodiment 50. The method of any one of Embodiments 1 to 49, wherein a total amount of the ISF collected from the insertion site occurs over a period ranging from 0.5 minutes to 1 hour, such as 1 minute to 15 minutes.

[0290] Embodiment 51. A system for collecting interstitial fluid (ISF) from a subject's skin, the system comprising: an energy source configured to deposit energy into a selected region of a subject's skin effective to facilitate ISF mobilization within the region; an array of microneedles configured to be inserted into the subject’s skin to form a plurality of micropores in the subject's skin in the selected region; a device configured to induce a pressure gradient, via positive pressure or suction, to further mobilize ISF and to withdraw ISF through the plurality of micropores formed by the array of microneedles; and means for collecting the ISF that has flowed from the micropores to the surface of the subject's skin.

[0291] Embodiment 52. A method for detection and / or monitoring of disease, injury, environmental exposure, or physiological or drug pharmacokinetic status in a subject, the method comprising: collecting interstitial fluid (ISF) from the skin of the subject in accordance with the method of any one of Embodiments 1 to 50; and analyzing the collected ISF for the presence or concentration of (i) a biomarker indicative of a specific disease injury, environmental exposure, or physiological status, (ii) a pharmaceutical compound, (iii) a metabolite, or (iv) a combination thereof. 46 53430309.117625-0344 GTRC 2024-159

[0292] Embodiment 53. The method of any one of Embodiments 1 to 50 or 52, wherein the array of microneedles is inserted into the subject’s skin, and rapidly removed from and reinserted into the subject’s skin two or more times.

[0293] Embodiment 54. The method of Embodiment 53, wherein the two or more sequential re-insertions of the array of microneedles occur within an interval ranging from 1 second to 1 minute in order to re-insert at least some of the microneedles into previously created micropores, such that the number of micropores is equal to less than the number microneedles in the array multiplied by the number of microneedle applications to the skin.

[0294] Embodiment 55. The method of Embodiment 54, wherein the number and frequency of re-insertions are effective to at least partially, or fully, provide the applied energy to facilitate interstitial fluid (ISF) mobilization to and / or within the selected region.

[0295] Embodiment 56. The method of any one of Embodiments 1 to 50 or 52 to 55, wherein each energy exposure creates a substantially uniform application of energy deposits within the selected region. 47 53430309.1

Claims

17625-0344 GTRC 2024-159 CLAIMS That which is claimed is:

1. A method comprising: applying energy into a selected region of a subject’s skin effective to facilitate interstitial fluid (ISF) mobilization to and / or within the selected region; inserting an array of microneedles into the subject’s skin at an insertion site to form a plurality of micropores in the subject’s skin in the selected region; and collecting ISF from the insertion site.

2. The method of claim 1, wherein the collecting ISF comprises collecting ISF that has flowed from the micropores onto the surface of the subject’s skin or into a fluidic system or reservoir positioned on the subject’s skin at the insertion site, optionally wherein the collected ISF contacts a sensor that makes a measurement of the composition or other property of the ISF.

3. The method of claim 1, wherein the collecting ISF comprises collecting ISF via the microneedles which remain inserted at the insertion site.

4. The method of claim 3, wherein the microneedles are hollow or porous.

5. The method of claim 3, wherein the microneedles comprise a hydrogel material, which is configured to swell with collected ISF.

6. The method of any one of claims 1 to 5, wherein the array of microneedles is configured to penetrate at least across the stratum corneum and the viable epidermis, and into the dermis.

7. The method of claim 6, wherein the array of microneedles is configured to penetrate into the papillary dermis.

8. The method of claim 6, wherein the array of microneedles is configured to penetrate into the reticular dermis.

9. The method of any one of claims 1 to 5, wherein the microneedles have a length from about 100 µm to about 2 mm, such as from 250 µm to 750 µm. 48 53430309.117625-0344 GTRC 2024-159 10. The method of any one of claims 1 to 5, wherein the array of microneedles comprises from 2 microneedles to 1000 microneedles, such as from 5 to 500 microneedles.

11. The method of any one of claims 1 to 5, wherein the array of microneedles is inserted into the subject’s skin, and then one or more times removed from the subject’s skin and reinserted into the subject’s skin in the selected region.

12. The method of claim 11, wherein sequential insertions of the array of microneedles occur within an interval ranging from 1 second to 1 minute to create at least twice as many micropores as the number of microneedles in the array.

13. The method of claim 11, wherein a sequential insertion of the array of microneedles occurs following an interval ranging from 10 minutes to 24 hours to create new micropores after at least some of the previously created micropores have closed and / or their ability to pass ISF therethrough has become degraded.

14. The method of any one of claims 1 to 5, wherein the array of microneedles remains inserted in the skin for a period ranging from 1 minute to 24 hours, such as 5 minutes to 30 minutes, or 30 minutes to 6 hours.

15. The method of any one of claims 1 to 5, wherein from 2 to 4000 micropores, such as from 20 to 500 micropores, are formed in the selected region of the subject’s skin.

16. The method of claim 15, wherein the micropores initially are between 10 µm and 1000 µm deep, and between 10 µm and 300 µm wide, or wherein the cross-sectional area of each micropore initially ranges from about 0.01 mm2to about 0.1 mm2.

17. The method of any one of claims 1 to 5, wherein the array of microneedles is no longer inserted in the skin at the time of the collection of ISF from the subject’s skin.

18. The method of any one of claims 1 to 5, wherein the array of microneedles remains within the skin during the collection of ISF from the subject’s skin.

19. The method of any one of claims 1 to 5, wherein the application of energy is effective to induce local edema within the selected region of the subject’s skin.

20. The method of any one of claims 1 to 5, wherein the total energy applied is in an amount effective to produce sustained edema lasting more than 15 minutes. 49 53430309.117625-0344 GTRC 2024-159 21. The method of any one of claims 1 to 5, wherein the selected region is in an area of the subject’s skin which is about 0.1 cm2to 100 cm2, such as from 1 cm2to 10 cm2.

22. The method of any one of claims 1 to 5, wherein the energy applied into the selected region is applied in one or more energy increments of from 1 microsecond to 10 minutes.

23. The method of any one of claims 1 to 5, wherein the energy applied into the selected region is applied in two or more energy exposures, such as from 2 to 2000 energy exposures, or from 5 to 100 energy exposures.

24. The method of claim 23, wherein the two or more energy exposures are applied over a period from 1 second to 1 day.

25. The method of any one of claims 1 to 5, wherein the energy is applied to the selected region from an energy source selected from a light source, such as an ultraviolet light source, a visible light source, an infrared light source, or a laser.

26. The method of claim 23, wherein between each energy exposure, the energy source is removed from and repositioned on the subject's skin in the selected region.

27. The method of claim 23, wherein each energy exposure creates a fractional array of energy deposits within the selected region.

28. The method of claim 27, wherein each of the fractional arrays of energy deposits are not directly overlapping.

29. The method of claim 23, wherein between each energy exposure, the energy source remains in place on the subject's skin in the selected region.

30. The method of claim 23, wherein each energy exposure creates a uniform array of energy deposits within the selected region.

31. The method of claim 25, wherein the energy source is a laser.

32. The method of claim 25, wherein the energy from the light source has a wavelength ranging from 100 nm to 2100 nm. 50 53430309.117625-0344 GTRC 2024-159 33. The method of claim 25, wherein the energy from the light source has a pulsing frequency ranging from 1 Hz and 30 Hz.

34. The method of claim 23, wherein each energy exposure has an energy output ranging from 0.1 J per cm2to 20 J per cm2of surface of skin in the selected region.

35. The method of any one of claims 1 to 5, wherein the energy is applied to the selected region by radiofrequency (RF) waves, by ultrasound, and / or by electrical current.

36. The method of any one of claims 1 to 5, wherein the energy is applied to the selected region by mechanical means.

37. The method of any one of claims 1 to 5, wherein the application of energy is effective to generate heat within the epidermis and / or the dermis of the subject’s skin.

38. The method of any one of claims 1 to 5, wherein the collecting ISF that flows from the micropores to the surface of the subject’s skin comprises inducing flow of ISF within the subject’s skin by the application of a pressure gradient to the subject’s skin.

39. The method of claim 38, wherein the pressure gradient comprises application of a positive pressure in a range from 10 kPa to 1500 kPa, such as from 100 kPa to 800 kPa.

40. The method of claim 38, wherein the pressure gradient comprises application of a negative pressure in a range from -5 kPa to -100 kPa, such as from -30 kPa to -80 kPa.

41. The method of claim 38, wherein the pressure gradient is applied to the periphery of the selected region.

42. The method of claim 38, wherein the pressure gradient is applied to the periphery of each of the micropores in the subject’s skin in the selected region.

43. The method of claim 38, wherein the pressure gradient is applied to substantially all of the selected region.

44. The method of claim 38, wherein the application of a pressure gradient comprises repeated intermittent application of positive or negative pressure, such as at frequency ranging from about 0.2 Hz to about 30 Hz. 51 53430309.117625-0344 GTRC 2024-159 45. The method of any one of claims 1 to 5, wherein the collecting ISF that flows from the micropores to the surface of the subject’s skin is carried out following a delay period after the steps of applying energy and the inserting the array of microneedles.

46. The method of claim 45, wherein the delay period is in a range from 1 minute to 24 hours, such as from 2 minutes to 10 minutes.

47. The method of claim 45, wherein the surface of the subject’s skin about the selected area is cleaned during the delay period.

48. The method of any one of claims 1 to 5, wherein the ISF collected is in an amount from 1 µl to 100 µl, such as 10 µl to 60 µl.

49. The method of any one of claims 1 to 5, wherein the average rate of ISF collection over a total collection period for the subject is between 0.1 µl / minute and 100 µl / minute, such as 1 µl / minute to 20 µl / minute.

50. The method of any one of claims 1 to 5, wherein a total amount of the ISF collected from the insertion site occurs over a period ranging from 0.5 minutes to 1 hour, such as 1 minute to 15 minutes.

51. A system for collecting interstitial fluid (ISF) from a subject's skin, the system comprising: an energy source configured to deposit energy into a selected region of a subject's skin effective to facilitate ISF mobilization within the region; an array of microneedles configured to be inserted into the subject’s skin to form a plurality of micropores in the subject's skin in the selected region; a device configured to induce a pressure gradient, via positive pressure or suction, to further mobilize ISF and to withdraw ISF through the plurality of micropores formed by the array of microneedles; and means for collecting the ISF that has flowed from the micropores to the surface of the subject's skin.

52. A method for detection and / or monitoring of disease, injury, environmental exposure, or physiological or drug pharmacokinetic status in a subject, the method comprising: collecting interstitial fluid (ISF) from the skin of the subject in accordance with the method of any one of claims 1 to 5; and 52 53430309.117625-0344 GTRC 2024-159 analyzing the collected ISF for the presence or concentration of (i) a biomarker indicative of a specific disease injury, environmental exposure, or physiological status, (ii) a pharmaceutical compound, (iii) a metabolite, or (iv) a combination thereof.

53. The method of any one of claims 1 to 5, wherein the array of microneedles is inserted into the subject’s skin, and rapidly removed from and reinserted into the subject’s skin two or more times.

54. The method of claim 53, wherein the two or more sequential re-insertions of the array of microneedles occur within an interval ranging from 1 second to 1 minute in order to re- insert at least some of the microneedles into previously created micropores, such that the number of micropores is equal to less than the number microneedles in the array multiplied by the number of microneedle applications to the skin.

55. The method of claim 54, wherein the number and frequency of re-insertions are effective to at least partially, or fully, provide the applied energy to facilitate interstitial fluid (ISF) mobilization to and / or within the selected region.

56. The method of any one of claims 1 to 5, wherein each energy exposure creates a substantially uniform application of energy deposits within the selected region. 53 53430309.1

Images