Microneedles, microneedle patches, methods of making and methods of using same

Abstract

A microneedle comprising a base and a tip remote from the base, the microneedle being formed from an anisotropic porous composition comprising a plurality of channels extending through the microneedle in a substantially uniform direction from a surface of the base toward an outer surface defined by the tip, and the plurality of channels are adapted to enable fluid to flow therein.

Description

Technical Field

[0001] The present invention relates to microneedles, microneedle patches, methods of making and using the same. More specifically, the present invention relates to microneedles and microneedle patches comprising a plurality of uniform channels for facilitating fluid flow therein, methods of making and using the same. Background Art

[0002] Microneedles (MNs) are minimized needle arrays and are well known in the art for various uses such as, for example, drug delivery to / through the dermis and other locations, delivery of cells, etc. Several MN platforms have been developed for delivery of cells.

[0003] Although porous microneedles exist in the prior art, such pores within the microneedles are generally random in size and direction, and are sponge-like, with pores that result in many dead ends and closed-cell structures, thus limiting the efficiency of drug or cell delivery. US 2016 / 158511 A1 (US'511), entitled "Fabrication of Phase-Transition microneedle patch" and published on June 9, 2016 to Jin, describes a porous MN and its manufacture. However, the MN in US'511 contains interconnected pores that are not irregularly oriented channels. In addition, it is believed that the diameters of these interconnected pores are not uniform, but are distributed within a range.

[0004] Further, previous methods for producing and / or loading microneedles with payloads typically employ centrifugation, a harsh and high-gravity process. Since certain cells and molecules to be delivered by MNs may be quite fragile, such centrifugation may damage and / or destroy certain cells if they are the intended payload.

[0005] Thus, there remains a need for microneedles that provide easier manufacturing, easier payload loading, reduced payload damage / inactivation, unidirectional flow for reducing potential cross contamination, provide anisotropic properties, and / or precisely deliver payloads to anisotropic porous MNs. In addition, it has been found that there is a need to provide a manufacturing and / or loading method for preparing cells for transdermal cell delivery that is substantially free of payload centrifugation. There also remains a need for a method and article for sampling fluids such as tears that does not rely on, for example, commercial tear test strips (Schirmerstrips) and / or hard materials such as capillary glass tubes. Summary of the invention

[0006] In a first aspect, a microneedle is provided, comprising a base and a tip distal to the base, wherein the microneedle is formed from an anisotropic porous composition, the anisotropic porous composition comprising a plurality of channels extending through the microneedle in a substantially uniform direction from a surface of the base toward an outer surface defined by the tip, and wherein the plurality of channels are adapted to enable fluid to flow therein.

[0007] Preferably, each channel of the plurality of channels has a substantially uniform channel diameter.

[0008] Preferably, the channel diameter is about 20 μm; or about 30 μm; or between 20-50 μm; or between 20-150 μm; or between 85-145 μm.

[0009] Preferably, the flow of fluid within the plurality of channels is unidirectional.

[0010] Preferably, the plurality of channels have a first physical property when fluid flows into the microneedles and have a second physical property when fluid flows out of the microneedles, and wherein the first physical property is different from the second physical property.

[0011] Preferably, the first physical property and the second physical property include morphological properties, mechanical properties, pore structure and Young's modulus.

[0012] Preferably, the anisotropic porous composition is formed from a cross-linked polymer matrix.

[0013] Preferably, the cross-linked polymer matrix comprises a plurality of monomers selected from the group consisting of gelatin, alginate, polyvinyl alcohol (PVA), poly(2-hydroxyethyl methacrylate) (PHEMA), polyacrylamide (PAAm), vinyl-modified hyaluronic acid (HA), methactylated hyaluronic acid (MeHA), and combinations thereof.

[0014] In a second aspect, a microneedle patch is provided, comprising a substrate and at least one microneedle disposed on the substrate.

[0015] Preferably, the substrate is integrally formed with the microneedles, and wherein the plurality of channels extend through both the substrate and the microneedles from an outer surface of the substrate toward an outer surface defined by the tips of the microneedles.

[0016] Preferably, the substrate is formed separately from the microneedles and is attached to the base of the microneedles.

[0017] Preferably, the substrate is a metal sheet.

[0018] Preferably, the substrate is a handle.

[0019] Preferably, the microneedle patch further comprises an electrochemical sensor attached to the substrate, and preferably, a payload pre-loaded within the microneedle patch.

[0020] Preferably, the payload is selected from the group consisting of cells, drugs, macromolecules, extracellular vesicles, and combinations thereof.

[0021] Preferably, the cell is selected from the group consisting of immune cells, antigen cells, stem cells, melanocytes, hair follicle cells, beta cells, fibroblasts, therapeutic cells, preventive cells and combinations thereof.

[0022] Extracellular vesicles (EVs) are cell-derived membrane-enclosed vesicles that carry biologically active molecules and deliver them to recipient cells. Preferably, the extracellular vesicles are selected from the group consisting of exosomes, microvesicles, apoptotic bodies, autophagic extracellular vesicles, matrix vesicles, stressed extracellular vesicles, and combinations thereof.

[0023] Preferably, the macromolecule is selected from the group consisting of genetic material, polypeptides, proteins, deoxyribonucleic acid sequences (DNA), ribonucleic acid sequences (RNA), enzymes, antibodies, and combinations thereof.

[0024] Preferably, the microneedle patch further comprises a plurality of microneedles, wherein the plurality of microneedles forms a microneedle array disposed on a substrate.

[0025] In a third aspect, a method for making a microneedle is provided, the method comprising the following steps: (i) casting a prepolymer solution comprising a plurality of monomers into a mold defined by at least one recess formed with a microneedle structure, wherein the microneedle structure comprises a base and a tip distal to the base; (ii) freezing the prepolymer solution with a temperature gradient across the mold; (iii) cross-linking the plurality of monomers to form a cross-linked polymer matrix; (iv) freeze-drying the cross-linked polymer matrix to form an anisotropic porous composition defined by the microneedle structure, wherein the anisotropic porous composition comprises a plurality of channels extending through the anisotropic porous composition in a substantially uniform direction from a surface of the base toward an outer surface defined by the tip; and (v) removing the anisotropic porous composition from the mold.

[0026] Preferably, the temperature gradient is provided by arranging a first surface of the mold at room temperature, or at a temperature between 4 and 28°C, or at a temperature between 18 and 24°C, and further arranging a second surface of the mold opposite to the first surface at a temperature between -0°C and -300°C, or at a temperature between -25°C and -285°C, or at a temperature between -50°C and -250°C.

[0027] Preferably, the second surface of the mold is placed in contact with a cooling element selected from the group consisting of: liquid nitrogen, liquid helium, dry ice and mixtures thereof.

[0028] Preferably, the cooling element is in the form of a cooling bath.

[0029] Preferably, cross-linking of the plurality of monomers is achieved by freezing the prepolymer solution at sub-zero temperature.

[0030] Preferably, cross-linking of the plurality of monomers is achieved by exposing the prepolymer solution to a UV source.

[0031] Preferably, the method further comprises the step of thawing the cross-linked polymer matrix at room temperature prior to the freeze-drying step (iv).

[0032] Preferably, the cross-linked polymer matrix is ​​thawed in deionized water for 1 hour to 3 days, or 4 hours to 2 days, or 6 hours to 36 hours.

[0033] Preferably, the mold is formed of a mold material selected from a first group consisting of: polymers, metals, and combinations thereof; or selected from a second group consisting of: polydimethylsiloxane (PDMS), steel, and combinations thereof.

[0034] Preferably, each channel of the plurality of channels has a substantially uniform channel diameter.

[0035] Preferably, the method further comprises the step of adjusting the channel diameter by varying the polymer concentration of the prepolymer solution.

[0036] Preferably, the method further comprises the step of adjusting the channel diameter by varying the temperature gradient.

[0037] Preferably, the method further comprises the step of adjusting the channel diameter to about 20 μm, or about 30 μm, or between 20-50 μm, or between 20-150 μm, or between 85-145 μm.

[0038] Preferably, the plurality of monomers are selected from the group consisting of gelatin, alginate, polyvinyl alcohol (PVA), poly(2-hydroxyethyl methacrylate) (PHEMA), polyacrylamide (PAAm), vinyl-modified hyaluronic acid (HA), methacrylated hyaluronic acid (MeHA), and combinations thereof.

[0039] In a fourth aspect, a method of manufacturing a microneedle patch is provided, the method comprising the steps of the microneedle manufacturing method, and further comprising the step of forming a substrate having at least one microneedle disposed thereon.

[0040] Preferably, the substrate is formed by casting the prepolymer solution into the mould beyond filling the recesses of the mould along the mould walls.

[0041] Preferably, the substrate is formed by attaching a separate layer to the base of the microneedles.

[0042] Preferably, the separate layer is a metal sheet.

[0043] Preferably, the separate layer is a handle.

[0044] Preferably, the method further comprises the step of loading the microneedle patch with a payload.

[0045] Preferably, the payload is selected from the group consisting of: cells, drugs, extracellular vesicles, macromolecules, and combinations thereof.

[0046] Preferably, the cell is selected from the group consisting of immune cells, antigen cells, stem cells, melanocytes, hair follicle cells, beta cells, therapeutic cells, fibroblasts, preventive cells and combinations thereof.

[0047] Preferably, the extracellular vesicle is selected from the group consisting of exosomes, microvesicles, apoptotic bodies, autophagic extracellular vesicles, matrix vesicles, stressed extracellular vesicles and combinations thereof.

[0048] Preferably, the macromolecule is selected from the group consisting of genetic material, polypeptides, proteins, deoxyribonucleic acid sequences (DNA), ribonucleic acid sequences (RNA), enzymes, antibodies, and combinations thereof.

[0049] Preferably, the payload is loaded into the prepolymer solution as the polymer solution is cast in the mold.

[0050] Preferably, the payload is loaded into the anisotropic porous composition, followed by the further step of attaching a substrate to the base of the microneedles.

[0051] Preferably, the method further comprises the step of attaching the electrochemical sensor to a substrate.

[0052] In a fifth aspect, a method of using a microneedle patch is provided, the method comprising the step of applying the microneedle patch to an application area to deliver a payload from the microneedle patch to the application area.

[0053] Preferably, the area of ​​application is the skin surface.

[0054] Preferably, the area of ​​application is the ocular surface.

[0055] Preferably, the area of ​​application is the oral cavity surface.

[0056] In a sixth aspect, a method for using a microneedle patch is provided, comprising the step of contacting a sample with the microneedle patch to collect the sample.

[0057] Preferably, the sample is tear fluid. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] In order to obtain a more accurate understanding of the above invention, the invention briefly described above will be described in more detail with reference to the specific embodiments of the invention briefly described above illustrated in the accompanying drawings. The drawings presented herein may not be drawn to scale, and any reference to size in the drawings or the following description is specific to the disclosed embodiments.

[0059] Figure 1 A schematic diagram showing an embodiment of a microneedle (MN) patch according to the present invention;

[0060] Figure 2 A partially cut-away side view of a MN patch according to an embodiment of the present invention is shown.

[0061] Figure 3 shows a close-up schematic diagram of an embodiment of a microneedle according to the present invention;

[0062] Figure 4 shows a schematic diagram of a mold when the mold according to an embodiment of the present invention is filled with a polymer solution;

[0063] Figure 5 Shown according to Figure 4 A schematic diagram of a filled mold;

[0064] Figure 6 FIG. 2 shows a frozen Figure 5 A schematic diagram of a filled mold similar to the filled mold in FIG.

[0065] Figure 7 A schematic diagram showing an embodiment of an array of MNs being removed from a mold;

[0066] Figure 8 A schematic diagram showing an embodiment of a manufacturing method according to the present invention;

[0067] Fig. 9 shows a schematic diagram of an embodiment of a manufacturing method according to the present invention, wherein a payload is added to a prepolymer solution while the prepolymer solution is in a mold;

[0068] Fig.10 A schematic diagram showing an embodiment of a method for using a MN patch according to the present invention;

[0069] Fig.11shows a schematic diagram of a monitoring system;

[0070] Fig.12 A schematic diagram showing an embodiment of a microneedle according to the present invention for transdermal cell delivery and tear fluid extraction;

[0071] Fig.13 a shows a schematic diagram of an embodiment of a manufacturing method according to the present invention;

[0072] Fig.13 b shows representative scanning electron microscopy (SEM) images of anisotropic porous cryogel (APC-), isotropic porous cryogel (IPC-), freeze-dried hydrogel (LH-) microneedles, and air-dried hydrogel (SH-) from the cross-section;

[0073] Fig.13 c is a schematic diagram showing a longitudinal sectional view and a cross-sectional view of a microneedle according to an embodiment of the present invention;

[0074] Fig.13 d shows the Fig.13 b Representative fluorescent images of longitudinal cross-sectional views of cryosectioned APC-, IPC-, and LH-MNs;

[0075] Fig.14a SEM images of APC-, IPC-, LH-, and SH-cylindrical materials viewed from a cross-section and from a longitudinal section are shown;

[0076] Fig.14b SEM images of APC-MNs with polymer concentrations of 2.5 wt.%, 5 wt.%, 7.5 wt.%, and 10 wt.%, produced using freezing temperatures of -20°C, -80°C, and -196°C, viewed from a longitudinal cross section are shown;

[0077] Fig.14c The flow process of the Cy3 dye solution from the needle tip to the substrate in APC-MNs with polymer concentrations of 2.5wt.%, 5wt.%, and 7.5wt.%; and the flow process from the needle tip to the substrate in IPC-MNs, LH-MNs, and solid hydrogels (SH-MDs) with a polymer concentration of 7.5wt.% are shown;

[0078] Fig.15 a shows a schematic diagram of an embodiment of the tear extraction process and post-analysis using APC-MNs according to the present invention;

[0079] Fig.15 b shows a graph indicating the results of mechanical testing of APC-MNs, where a horizontal force (shear force) was applied to the top of the MNs;

[0080] Fig.15 c shows hematoxylin and eosin (H&E) staining images of untreated mouse eyes and mouse eyes treated with APC-MNs and SH-MNs;

[0081] Fig.15 d shows a graph indicating the percentage of FITC-dextran (10 kDa) recovered from MN, commercial tear test strips, and cotton swabs, respectively;

[0082] Fig.15 e and 15f show graphs indicating (e) recovery and (f) IFN-γ from APC-MNs, commercial test strips, and swabs at a polymer concentration of 5 wt.%, respectively;

[0083] Fig.16 a is a schematic diagram showing an embodiment of a method for manufacturing APC-ice microneedles (cryoMNs) according to the present invention;

[0084] Fig.16 b shows optical images indicating the water contact angles on the PDMS negative microneedle molds including an unfilled (untreated) mold, an unfilled but hydrophilically treated (plasma treated) mold, and a mold filled with APC-MNs (Filled with APC-MNs), which were used to measure the water contact angles, respectively.

[0085] Fig.16 c shows a graph showing the quantitative water contact angles on the PDMS negative microneedle molds including an unfilled (Untreated) mold, an unfilled but hydrophilically treated (Plasma Treated) mold, and a mold filled with APC-MNs (Filled with APC-MNs);

[0086] Fig.16 d shows an image of the Cy3 dye distribution in the needle tip holes of the PDMS negative microneedle mold including an unfilled (untreated) mold, an unfilled but hydrophilic-treated (plasma-treated) mold, and a mold filled with APC-MNs (filled with APC-MNs);

[0087] Fig.16 e shows a graph indicating the fluorescence distribution within the PDMS negative microneedle mold including an unfilled (untreated) mold, an unfilled but hydrophilically treated (plasma treated) mold, and a mold filled with APC-MNs (filled with APC-MNs);

[0088] Fig.16 f shows an optical image showing the morphology of APC-cryomicroneedles (APC-cryoMNs);

[0089] Fig.16g shows a close-up image of the oriented porous structure of APC-cryoMN after melting in PBS;

[0090] Fig.16 h shows a statistical graph showing the water contact angle of the PDMS negative microneedle mold including an unfilled (untreated) mold, an unfilled but hydrophilically treated (plasma treated) mold, and a mold filled with APC-MNs (filled with APC-MNs);

[0091] Fig.17a The distribution of fluorescent dyes with different molecular weights (FITC-dextran and Cy3 with molecular weights of 10 kDa and 150 kDa or FD1W and FD15W) in the tips of APC2.5-MN, APC5-MN, IPC5-MN, LH5-MN, and APC7.5-MN left in the PDMS mold is shown.

[0092] Fig.17b The distribution of FITC-LNPs in the needle tips of APC2.5-MN, APC5-MN, IPC5-MN, and APC7.5-MN remaining in the PDMS mold is shown;

[0093] Fig.18 Photographic images of demolded APC-cryoMNs whose bases were made of various base formulations including PBS, PBS containing 10 wt.% 10 kDa HA, and PBS with 10 wt.% 13 kDa PVA are shown;

[0094] Fig.19 The distribution of hMSC, DC, 3T3, B16, and C2C12 cell infiltration in the APC-MNs retained in the PDMS mold is shown;

[0095] Fig. 20 a and Fig. 20 b shows an optical image and a thermal imaging image of a microneedle patch according to an embodiment of the present invention, wherein the microneedle patch is in a thawing process at room temperature;

[0096] Fig. 20 c shows the display Fig. 20 b is a graph showing the temperature of the microneedle patch changing with time;

[0097] Fig. 20 d shows Fig. 20 a and Fig. 20 b) a thermal imaging image and an optical image of a microneedle patch, wherein the microneedle patch is in the process of thawing at skin room temperature;

[0098] Fig. 20 e shows the display Fig. 20d is a graph showing the temperature variation of the microneedle patch over time;

[0099] Fig. 20 f shows a graph showing the compression test results of cyroMN (pure cryoprotectant), APC2.5-cryoMN, APC5-cryoMN and APC7.5-cryoMN at a displacement speed of 0.5 mm / s;

[0100] Fig. 20 g shows histological H&E images of pig skin treated with APC5-cryoMN and untreated pig skin;

[0101] Fig.21 a shows the distribution of γδ T cells in the needle tips of APC2.5-MN, APC5-MN, IPC5-MN, and APC7.5-MN when the cells were loaded onto APC-MNs remaining in the PDMS mold;

[0102] Fig.21 b shows the tip of the detached microneedle loaded with cells;

[0103] Fig.21 c and Fig.21 d shows a graph indicating the cell viability of γδ T cells cryopreserved in (c) cryovials and (d) APC-MNs;

[0104] Fig.21 e shows a graph indicating the percentage of γδ T cells positive for T cell receptor Vδ2 after recovery from APC-cryoMNs;

[0105] Fig. 22 a shows an optical image of the posterior skin of a mouse to which APC-cryoMNs were administered;

[0106] Fig. 22 b shows the CD3 in spleen, lung, liver, and tumor of mice 3 days after γδ T cell injection. + Vδ2 + Representative flow cytometry plots of cells;

[0107] Fig. 22 c shows the display of CD3 + Vδ2 + Graph of flow cytometric analysis of cell frequency in mouse spleen, lung, liver, and tumor;

[0108] Fig. 22 d shows Vδ2 in mouse skin tissue + Representative immunostaining images of cells;

[0109] Fig.23a shows the luciferase activity of MSTO-luc mice in each treatment group during the MSTO-luc mouse experiment;

[0110] Fig.23 b shows the Fig.23 a Body weight changes of MSTO-luc mice in each treatment group during the experimental period;

[0111] Fig.24 a shows the timeline of experiments involving NSG mice bearing MSTO-luc;

[0112] Fig.24 b shows representative images of luciferase activity in MSTO-luc-bearing mice that received Vδ2 T cells via APC-cryoMN or intravenous (iv) route in a 1- or 2-dose schedule;

[0113] Fig.24 c shows a graph indicating tumor growth measured by luciferase activity over time compared to day 2 after MSTO-luc tumor injection for different treatment groups;

[0114] Fig.24 d shows the survival curves of MSTO-luc mice in different treatment groups over time;

[0115] Fig.25 a shows the timeline of experiments involving A375-bearing NSG mice;

[0116] Fig.25 b shows a photographic representation of tumor volumes in mice injected with PBS as a control and mice treated with Vδ2 T cells by iv and APC-cryoMN routes over a period of 11 days;

[0117] Fig.25 c shows a statistical graph indicating the tumor volume measured from mice from each treatment group over time;

[0118] Fig.25 d shows a graph showing the changes in body weight of mice in each treatment group over time; and

[0119] Fig.25 e shows the expression of Vδ2 in melanoma tumor tissue and skin tissue + Representative immunostaining images of cells. DETAILED DESCRIPTION

[0120] 1. Definition

[0121] Unless expressly stated otherwise, all tests herein were conducted under standard conditions, which included test temperatures at room temperature and 25°C, sea level (1 atm.) pressure, pH 7, and all measurements were in metric units. In addition, all percentages, ratios, etc. herein are by weight unless expressly indicated otherwise. It is to be understood that, unless expressly stated otherwise, the material compounds, chemicals, etc. described herein are generally commercial and / or industrial standards that can be obtained from many suppliers around the world.

[0122] As used herein, the term "anisotropic" indicates that the physical properties of the porous microneedles vary depending on the direction in which the material travels through the channels in the microneedles. Examples of anisotropic properties herein include, for example, morphological properties, mechanical properties, pore structure, and / or Young's modulus.

[0123] As used herein, the term "loaded" indicates that a payload is contained within the channels of anisotropic porous MNs for delivery to a target. In contrast, the term "unloaded" as used herein indicates that no payload is contained within the channels of anisotropic porous MNs. Unless otherwise specifically indicated, these terms generally refer to a specific anisotropic porous MN.

[0124] As used herein, the term "lyophilization" indicates a process comprising the steps of: 1) freezing an item at atmospheric pressure in a chamber, the item containing a liquid, typically water; and 2) reducing the chamber pressure to allow the liquid to sublime from the frozen matrix, thereby leaving the matrix structure intact. As used herein, the term "lyophilized" describes an item that has undergone a lyophilization process. In addition, as used herein, the term lyophilization includes the phrase "freeze drying".

[0125] As used herein, the term "microneedle" or "MN" refers to a microscopic needle. Such needles may have a height of about 25 μm to about 2000 μm. They are typically in the form of an array or patch and may be made of a variety of materials.

[0126] As used herein, the term "needle length" refers to the length from the base of a microneedle to the needle tip of the microneedle.

[0127] As used herein, "unidirectional flow" refers to flow that travels in only one direction when delivering through the microneedle tip end or when extracting a sample through the microneedle tip end.

[0128] 2. Microneedle patch of the present invention

[0129] In embodiments of the present invention, anisotropic porous microneedles (MNs) contain a channel, or multiple channels, located therein. The channel or channels provide anisotropic properties. In embodiments herein, anisotropic porous MNs are absorbent anisotropic porous MNs, or absorbent anisotropic porous MNs that collect tear fluid, or anisotropic porous MNs that load cells. Without wishing to be limited by theory, it is believed that such anisotropic porous microneedles can be used, for example, for drug delivery, liquid sample extraction, cell delivery, and the like.

[0130] Figure 1 is a schematic diagram of an embodiment of a microneedle patch 10 according to the present invention. The MN patch 10 contains a plurality of anisotropic porous MNs 12 formed of a polymer matrix 14. Each of the anisotropic porous MNs 12 includes a microneedle tip 16 located at the distal end of a microneedle base 18. The microneedle base 18 is integral with a substrate 20.

[0131] Without wishing to be bound by theory, it is believed that although previous MNs have pores, such pores are generally random in size, direction, and are like sponges with pores resulting in many dead ends and closed-cell structures. In contrast, it is believed that the channels of the present invention are significantly different from pores in that they have diameter, length, and direction; or a significantly uniform diameter, significantly uniform length, significantly uniform direction, and combinations thereof. In addition, it is believed that the channels herein can be anisotropic in that the physical properties (e.g., morphological properties and mechanical properties) of the channels are different when the liquid therein flows in different directions. Anisotropic channels in MNs can be formed by the manufacturing method described herein, which relies on ice crystal growth during unidirectional freezing. In addition, it is believed that the material flow in the channels herein can be unidirectional.

[0132] In embodiments herein, the channel has a channel diameter, and this channel diameter is substantially uniform, meaning that the channel diameter varies less than about ±25%, or from about 0% to about 25%, or from about 3% to about ±15%, or from about 5% to about 15% along the length of the channel. Without wishing to be limited by theory, it is believed that uniform channel diameter provides benefits such as, for example, greater predictability, more consistent loading, consistent and predictable extraction and / or loading speeds, batch-to-batch consistency, etc.

[0133] Figure 2 FIG. 1 shows a partially cut-away side view of a MN patch 10 according to an embodiment of the present invention. Figure 2, the MN patch is cross-sectionally cut in the longitudinal direction, thereby showing that the MN 12 contains a plurality of channels 22 located therein. These channels 22 extend from the substrate layer 20 to the MN base 18 and to the MN tip 16. Due to the anisotropic structure of the microneedle patch 10, the channels extend uniformly from the base of the substrate 20 to the tip of the microneedle 16 in a direction perpendicular to the substrate layer 20.

[0134] Without wishing to be limited by theory, it is believed that the MNs herein provide a variety of potential benefits over previous MNs, such as, for example, unidirectional flow into or out of the MN, easier loading of payloads (i.e., materials or compositions to be delivered) into the MN, easier collection of samples (e.g., biological fluids), reduced irritation and tissue damage during sample collection, channel size control / channel size adjustability, production of MNs that can be used in a variety of different situations using a single manufacturing process, uniform channel size, and combinations thereof.

[0135] In embodiments herein, the anisotropic porous microneedles comprise a cross-linked polymer matrix; or are formed by a cross-linked polymer matrix. The cross-linked polymer matrix forms a channel; or defines the channel edge and provides a unidirectional flow via, for example, capillary action. In embodiments herein, the cross-linked polymer matrix contains monomers selected from the group consisting of: for example, gelatin, polyvinyl alcohol (PVA), polymethacrylate 2-hydroxyethyl ester (PHEMA), polyacrylamide (PAAm), vinyl-modified hyaluronic acid (HA), and combinations thereof; or gelatin, alginate, and combinations thereof. Typically, the cross-linked polymer matrix is ​​formed by a prepolymer solution as described herein.

[0136] The cross-linked polymer matrix herein contains one channel, or multiple channels, or channels with substantially uniform diameters, or channels with uniform diameters. The channels, or channel edges, are defined by the edges of the polymer matrix. Without wishing to be limited by theory, it is believed that channels with uniform diameters provide significant benefits, such as, for example, uniform payload delivery, uniform sample collection, a soft and / or non-irritating feel to, for example, the surface of the eye, a location for extracting samples, and the like. Furthermore, without wishing to be limited by theory, it is believed that the anisotropic porous MNs herein may be able to absorb larger volumes of liquids than comparable previous MNs. Furthermore, it is believed that the volume of the anisotropic porous MNs may not increase; or may remain substantially constant even after absorbing the sample, as well as combinations of the two.

[0137] Figure 3A close-up schematic diagram of an embodiment of an anisotropic porous MN 12 is shown. The anisotropic porous MN 12 is formed of a polymer matrix 14 that defines channels 22 therein. The channels have a channel diameter D that is substantially uniform along the length of the channels 22 in the anisotropic porous MN 12.

[0138] Typically, the channel diameter useful herein is greater than about 20 μm, greater than about 30 μm, or from about 20 μm to about 150 μm, or from about 85 μm to about 145 μm. In embodiments herein, the channel diameter is from about 20 μm to about 50 μm, especially when the anisotropic porous MN is used for drug delivery (i.e., the payload is a drug, especially a small molecule drug). Alternatively, if the payload to be delivered is a cell, in embodiments herein, the channel diameter is from about 85 μm to about 145 μm, depending on the size of the cells to be delivered.

[0139] The cells used as payloads herein can be, for example, immune cells, antigen cells, stem cells, fibroblasts, melanocytes, hair follicle cells, β cells, and combinations thereof. In embodiments herein, the cells are therapeutic cells, preventive cells, and combinations thereof. The cells herein can be human cells, animal cells, plant cells, chimeric cells; or human cells, cells of human origin, and combinations thereof. Since the diameter of human cells is typically from about 5 μm to about 30 μm, when the payload contains such human cells, the channel size should be at least about 30 μm to form a clear path for cells to move from the channel to the outside of the anisotropic porous MN.

[0140] Alternatively, the payload herein may include cells, drugs, macromolecules, and combinations thereof; or genetic material, bacteria, viruses, animal cells, cell extracts, and combinations thereof; or proteins, DNA, RNA, EVs, human cells, animals, non-human cells, chimeric cells, vectors, and combinations thereof; or cells; or drugs. Macromolecules may be genetic material, polypeptides, and combinations thereof; or proteins, deoxyribonucleic acid sequences (DNA), ribonucleic acid sequences (RNA), or combinations thereof; or enzymes, antibodies, and combinations thereof.

[0141] Furthermore, it is believed that by preloading the MNs with a payload and then lyophilizing the (preloaded) MNs, this can protect the MNs and the payload from adverse effects during transportation, storage, etc., such as structural collapse, cell inactivation, loss of efficacy, denaturation, degradation, etc.

[0142] In embodiments herein, the microneedle array contains a plurality of MNs, or about 1 MN to about 1000 MNs, or about 10 MNs to about 500 MNs, or about 100 MNs to about 250 MNs, or about 1 MN to about 100 MNs. In embodiments herein, the density of MNs in the microneedle array is determined by the size of the MNs. Typically, the density of MNs is about 1 MN / cm to about 20 MN / cm. Such MNs are typically formed together into an integrated MN array.

[0143] Without wishing to be bound by theory, it is believed that the anisotropic porous MNs herein or MN arrays herein may have great flexibility and may be applied to many different uses. For example, if the anisotropic porous MNs herein are pre-loaded with a payload, the anisotropic porous MNs may provide consistent payload delivery, or when not pre-loaded with a payload, it may provide consistent sample absorption and / or efficient retrieval of microliter amounts.

[0144] 3. Manufacturing method

[0145] In the embodiments of the present invention, anisotropic porous MNs can be formed by a manufacturing method comprising the following steps: providing a prepolymer solution containing a plurality of monomers therein; providing a mold containing a mold top surface and a mold bottom opposite to the mold top surface; casting the prepolymer solution into the mold; providing a temperature gradient between the mold top surface and the mold bottom; freezing the prepolymer solution using the temperature gradient; cross-linking the plurality of monomers to form a cross-linked polymer matrix in the mold; and freeze-drying the MN scaffold to form anisotropic porous MNs.

[0146] Figure 4 Schematic diagram of a mold 30 according to an embodiment of the present invention is shown. The mold 30 includes a mold top surface 32, a mold wall 34 and a plurality of recesses 36, which are used to form a negative impression of the MN 12 (see Figure 1 ). In addition, an applicator 38 is shown which applies a prepolymer solution 40 into the mold 30, thereby casting the prepolymer solution 40 into the mold. It can be seen that in this case, the mold wall 34 defines the perimeter of the mold 30, thereby allowing the mold 30 to be deeper than the depth of the recess 36. This allows the base layer to be formed in the following manner (see Figure 1 20): Fill the mold 30 beyond the point where the recess 36 is filled and ensure that the base layer (see Figure 1 20) and MN (see Figure 1 12) are one.

[0147] Reference now Figure 5 , which shows Figure 4Schematic diagram of a filled mold 30. Once the mold 30 is filled with the prepolymer solution 40, the mold bottom 42 is secured to the mold 30 opposite the mold top surface 32. The outer surface of the base 42 forms a watertight seal with the mold wall 34. Figure 5 , it can be seen that the prepolymer solution 40 fills the recess 36 and the mold area adjacent to the mold wall 34. This ensures that there is enough prepolymer solution 40 to form the base layer 20 in its entirety.

[0148] Figure 6 FIG. 2 shows a frozen Figure 4 Schematic diagram of an embodiment of a filled mold 30 similar to the filled mold in FIG. The filled mold 30 is relative to Figure 4 Inverted, and the mold bottom 42 is placed on a cooling element 50. The substrate 20 is sealed by the mold bottom 42 (which in this case is a tin foil) to prevent the prepolymer solution 40 from leaking out of the mold 30. The mold top surface 32 is in contact with air at room temperature. Therefore, the mold top surface 32 and the mold bottom 42 are subject to a temperature gradient 52.

[0149] The temperature gradient is formed by the difference between the temperature of the top surface of the mold and the temperature of the bottom of the mold. In embodiments herein, the top surface of the mold is at room temperature, or about 4°C to about 28°C, or about 18°C ​​to about 24°C. In embodiments herein, the bottom temperature of the mold is about -0°C to about -300°C, or about -10°C to about -285°C, or about -15°C to about -250°C. For example, if the top surface of the mold is at room temperature of 25°C and the bottom of the mold is on a cooling plate in liquid nitrogen at -196°C, the temperature gradient is 221°C.

[0150] Figure 7 A schematic diagram is shown of the MN patch 10 being removed from the mold 30. After curing and before or after loading, the MN patch 10 is removed from the mold 30 to release the MN 12 attached to and integral with the base layer 20.

[0151] Typically, the mold herein is a negative mold, which is typically formed of a flexible, low temperature resistant material such as a polymer, metal, and combinations thereof, or polydimethylsiloxane (PDMS), steel, copper, resin, and combinations thereof.

[0152] Embodiments of the methods herein further comprise the optional step of removing the anisotropic porous MN from the mold.In embodiments of the present invention, the anisotropic porous MN, or a MN array comprising a plurality of anisotropic porous MNs, is simply peeled off from the mold.

[0153] Figure 8A schematic diagram of an embodiment of a manufacturing method 70 according to the present invention is shown. In step 1, a prepolymer solution 40 is provided in an applicator 38, which in this case is a plastic or glass dropper. A mold 30 having a mold top surface 32 and an open mold bottom 42 is provided. The applicator 38 casts the prepolymer solution 40 onto the mold 30 by applying the prepolymer solution 40 to the mold. The mold 30 is centrifuged at 4,000 rpm for 3 min with the recess 36 pointing outward to remove any particles that may be present and thus block the MN tip (see Figure 1 16) to remove any bubbles formed. This produces a filled mold 30, as shown in step 2.

[0154] In step 3, a base layer 20 (formed of tin foil in this case) is added to the mold bottom 42 to seal the prepolymer solution 40 into the mold 30. In step 4, the mold 30 is inverted so that the mold bottom 42 is placed directly onto the cooling element 50, which contains liquid nitrogen at about -196° C. The mold top surface 32 is at room temperature of 25° C., thereby providing a temperature gradient of 225° C. This results in ice crystal channels extending unidirectionally from the mold bottom 42 toward the mold top surface 32.

[0155] In step 5, the prepolymer solution 40, still in the mold 30, is cross-linked in a sub-zero freezer for at least 1 day, or about 1 day to about 1 week, or about 1.5 days to about 4 days, so as to maintain the formed ice crystal channels and polymerize the monomers of the prepolymer solution to form the polymer matrix 14. Alternatively, cross-linking can be initiated by, for example, exposing the mold containing the frozen prepolymer solution to a UV source.

[0156] In step 6, the mold 30, now containing the polymer matrix 14, is thawed in, for example, deionized water at room temperature; or thawed in deionized water for about 1 hour to about 3 days, or about 4 hours to about 2 days, or about 6 hours to about 36 hours. Without wishing to be bound by theory, it is believed that this extended thawing process in deionized water also removes any unreacted compounds from the polymer matrix.

[0157] In step 7, the mold 30 is placed in a freeze dryer 72 with the mold top surface 32 facing downward and freeze dried to form the MN array 10, which contains anisotropic porous MNs 12, which further contain multiple channels 22. In step 8, the MN array 10 is demolded from the mold 30 by pulling the base layer 20.

[0158] Typically, the cross-linked polymer matrix is ​​formed by cross-linking of the ingredients in the prepolymer solution as described herein. In embodiments herein, the prepolymer solution is in the form of an aqueous solution, a hydrogel, and combinations thereof; or a cryogel. Prepolymer solutions useful herein contain, for example, monomers, catalysts, free radical initiators, and combinations thereof. The monomers herein may be polymer monomers, polymer subunits, and combinations thereof. In embodiments herein, the polymerization reaction is carried out via low temperature cross-linking, free radical polymerization initiated by, for example, light treatment (such as UV light), and combinations thereof. In embodiments herein, the low temperature cross-linking process comprises the following steps: phase separation, cross-linking, polymerization, and thawing via ice crystal growth formation to form a cryogel network having channels therein.

[0159] In embodiments herein, the prepolymer solution further contains monomers. As described herein, the prepolymer solution contains monomers cross-linked therein to form a cross-linked polymer matrix; or polymer monomers, polymer subunits and combinations thereof. In embodiments herein, the monomer is selected from gelatin, polyvinyl alcohol (PVA), vinyl-modified hyaluronic acid (HA), poly (ethylene glycol) diacrylate (PEGDA) and combinations thereof; or gelatin, alginate, methacrylated hyaluronic acid (MeHA) and combinations thereof. Useful monomers herein can be easily obtained from multiple suppliers worldwide.

[0160] Without wishing to be bound by theory, it is believed that when the prepolymer solution is frozen in the absence of a temperature gradient as described herein, only pores, or interconnected pores, are formed, and no directional channels are formed because the direction and size of the ice crystal growth is random.

[0161] Furthermore, in the previous MN, the monomers of the prepolymer solution may be cross-linked prior to freezing. In such a process, it is believed that the porous structure of the polymer is again non-oriented and traps the water (or any solvent being used) and other prepolymer solution components therein in pores of substantially random orientation, size, shape, etc.

[0162] Rather, and without wishing to be limited by theory, it is believed that the uniform channels in the present invention are a result of the present invention's controlled freezing step in the presence of a temperature gradient, which occurs prior to the crosslinking step. It is believed that freezing of the prepolymer solution in the presence of a temperature gradient causes ice crystals to form ice crystal channels having substantially uniform, or uniform, diameters. Specifically, at a molecular level, the thermodynamics of ice crystal formation pushes the monomers to the sides of the ice crystals. The monomers form with a specific and parallel orientation, and the diameters of the ice crystals are substantially uniform.

[0163] After freezing the prepolymer solution using a temperature gradient, the monomers or polymer monomers, polymer subunits are then cross-linked to form a cross-linked polymer matrix within the mold. Thus, when the mold and its frozen contents are subjected to freeze-drying, ice from the prepolymer solution; or aqueous solution, hydrogel and combinations thereof; or cryogel sublimes, leaving behind (hollow) channels formed and defined by the cross-linked polymer matrix. The resulting channels typically have a substantially uniform diameter.

[0164] Furthermore, it is believed that by adjusting the temperature of the bottom mould surface (ie, this results in a temperature gradient), the channels can be tuned to a particular diameter.

[0165] In embodiments herein, the anisotropic porous MNs in the MN patch are connected together by a base layer formed during the freezing step (which is generally formed of the same cross-linked polymer matrix as the anisotropic porous MNs), a cross-linked polymer matrix different from the anisotropic porous MNs, a handle, and combinations thereof while the MN array is still in the mold. In embodiments herein, when the base layer is a thin flexible material, it can be a commonly available thin flexible material, such as tin foil (also known as aluminum foil) and combinations thereof. Without wishing to be limited by theory, it is believed that tin foil may be useful herein because it is readily available, cheap, tough, flexible, and provides rapid heat transfer, etc.

[0166] A handle useful herein can be a 3D printed flat base with a stem of the appropriate size and shape for a hand to hold. The base is attached to the MN array, and the stem can be held in the user's hand.

[0167] In embodiments herein, the handle is formed of, for example, plastic and can be manufactured using a 3D printer. In embodiments herein, the handle is made of VeroClear available from Stratasys, Inc., Eden Prairie, Minnesota, USA. TM Formed using fused deposition manufacturing (a type of 3D printing). As desired, the support handle can be used instead of the thin flexible material, or can be used in addition to such thin flexible material. In embodiments herein, the support handle provides a heat sink that reduces the chance of the frozen MN melting before being administered or used to deliver a payload.

[0168] Fig. 9A schematic diagram of a further embodiment of the manufacturing method herein is shown. In step 1, a payload 54 is added to the prepolymer solution 40 while the prepolymer solution 40 is in the mold 30. The mold 30 thus contains the mixed payload 54 and prepolymer solution 40, and is subjected to a freezing step 56 and a cross-linking step (not shown) to form a polymer matrix 14 that already contains the payload 54. After the cross-linking step, a handle 58 is applied to the MN array 10 formed of the polymer matrix 14. The handle 58 is used to pull the MN array 10 formed with the anisotropic porous structure out of the mold 30 while still attached to the handle 58.

[0169] In one embodiment herein, the loading step occurs after freeze drying and while the anisotropic porous MN is still in the mold but without a substrate layer. Alternatively, in another embodiment herein, the loading step may occur after the prepolymer solution is added to the mold and before the freezing step. In either embodiment, the payload may be provided as a payload solution and added to the prepolymer solution and / or the anisotropic porous MN, for example dropwise, while the prepolymer solution and / or the anisotropic porous MN are still in the mold. Without wishing to be limited by theory, it is believed that such methods may reduce potential waste of payload in a backing layer, especially when the backing layer is formed of a polymer or linked monomers such as those found in a prepolymer solution. This benefit may be particularly important when the payload is scarce and / or expensive, such as in the case of drugs and autologous cells.

[0170] In embodiments herein, a payload solution may be applied to the polymer solution and / or the formed anisotropic porous MN prior to adding the substrate layer. If an anisotropic porous MN has already been formed, the payload may then be drawn in and / or distributed throughout the MN tip via, for example, capillary action. If the payload solution is added prior to the formation of the anisotropic porous MN (such as, for example, prior to a freezing stage when a prepolymer solution is present), the payload solution may mix with the prepolymer solution. Without wishing to be limited by theory, it is believed that such a process may increase payload loading efficiency and / or reduce payload waste in the substrate layer.

[0171] In the embodiments of the manufacturing methods herein, the loading step occurs when the payload (e.g., in the form of a payload solution) is loaded into the anisotropic porous MN before the anisotropic porous MN is removed from the mold. In the embodiments herein, the loading step is after the freeze-drying step. In such embodiments, the loading step can occur in the following ways: for example, the freeze-dried anisotropic porous MN (or an MN array containing anisotropic porous MNs, etc.) is immersed in the payload solution, the payload solution is sprayed onto the mold containing the anisotropic porous MNs, and combinations thereof. Without wishing to be limited by theory, it is believed that even if the payload solution is added to the substrate layer, the channel will guide the payload to the channel in the tip of the anisotropic porous MN, so that the payload can be effectively and evenly delivered.

[0172] In embodiments herein, the loading step is performed after the anisotropic porous MN is removed from the mold. For example, after freeze drying, the anisotropic porous MN is removed from the mold and contacted with a payload solution containing the payload. The payload solution is absorbed into the anisotropic porous MN via capillary action.

[0173] In embodiments herein, after the loading step, the anisotropic porous MN may be removed from the mold.

[0174] In embodiments herein, anisotropic porous MNs, MN arrays, monitoring systems, and / or fluid sampling systems are manufactured in the absence of a centrifugation step performed when an optional payload is contained therein, or a step of centrifuging the mold when the mold contains a payload. Without wishing to be limited by theory, it is believed that, compared to previously known methods, such a centrifugation step is not required to fill the channels with the payload because the channels are not a mixture of closed and open pores as in previous cryoMNs. Advantages of avoiding a centrifugation step may include avoiding cell damage, allowing a wider diversity of potential payloads and uniform distribution and consistent loading of drugs, reducing manufacturing complexity, reducing the expertise required to load anisotropic porous MNs, etc. Because cells and proteins can be particularly susceptible to damage by high shear and / or high centrifugal forces, the lack of such a centrifugation step when loading the payload is particularly useful when the payload contains cells, macromolecules, and combinations thereof.

[0175] The cooling element herein can be, for example, a highly refrigerated cooling plate, a container of ultra-low temperature liquids such as, for example, liquid nitrogen (bp -196°C), liquid helium (bp, -269°C), dry ice (mp, -78.5°C), and mixtures thereof. In embodiments herein, the cooling element is in the form of a cooling bath.

[0176] 4. Examples

[0177] 4.1 Example 1

[0178] In embodiments herein, the prepolymer solution contains 5% w / v gelatin (Sigma-Aldrich, St. Louis, MO, USA), 1 M 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) (Energy Chemical, Shanghai, China), and 1 M N-hydroxysuccinimide (NHS) (Macklin, Shanghai, China) in aqueous solution.

[0179] Provide as in Figure 4 Figure 1 shows a PDMS negative mold as seen in Figure 1, which contains a mold top surface and a mold bottom opposite the mold top surface. The mold bottom is open to the air. The mold contains a well that narrows toward the mold top surface to form the MN tip.

[0180] The prepolymer solution was cast into the mold by adding the prepolymer solution to the top surface of the mold and filling the voids with the prepolymer solution via centrifugation at 4,000 rpm. Excess prepolymer solution was added to the mold to form a backing layer. Tin foil was then added to the bottom of the mold to cover the wells and form a backing layer.

[0181] The room temperature was 25°C. The bottom of the mold was placed on a cooling plate cooled to a temperature of -20°C to provide a temperature gradient of 45°C for 10 minutes. After 10 minutes, the prepolymer solution was frozen solid and the mold containing the frozen prepolymer solution was stored in a -20°C freezer for two days. The polymer matrix was reacted with EDC and NHS to form the polymer matrix. The polymer matrix, still in the mold, was then thawed in deionized water for 1 day to remove unreacted chemicals.

[0182] The mold containing the thawed polymer matrix was placed in a freeze dryer (NingboScientz Biotechnology CO., LTD, Ningbo, China, SCIENTZ-10N) cooled to -80°C for complete pre-freezing for 2 hours. The freeze dryer was sealed and evacuated, and the freeze drying process was started. The process was completed within 4 hours, thereby obtaining a MN array containing anisotropic porous MNs in the PDMS mold.

[0183] The mold containing the anisotropic porous MNs was removed from the freeze dryer and stored in a drying oven at room temperature. The MN array contained a 10×10 array of anisotropic porous MNs, each MN having a needle height of about 850 μm, a square base (each side of the square was about 260 μm), and a backing layer thickness of about 1 mm. The pore diameter was about 100 μm as measured using an optical microscope and confirmed by SEM measurements. The MN array was peeled off from the mold by pulling the backing layer, thereby obtaining a MN array containing a plurality of anisotropic porous MNs.

[0184] 4.2 Example 2

[0185] MN arrays were fabricated according to the process described in Example 1, except that the MN arrays were not removed from the mold and formed without a backing layer. A payload of T cells (isolated from peripheral blood mononuclear cells) suspended in PBS cryoprotectant containing 2.5 wt.% DMSO and 100 mM sucrose was prepared. 30 μL of payload was added to the bottom of the mold to form a loaded MN array without centrifuging the payload. A handle was applied to cover all MN bases, replacing the basal layer. The entire device (as in Fig. 9 The mold, prepolymer solution, handle, etc. (see Figure 2) is frozen in a standard gradient cryopreservation process (-1°C / min) such as using a gradient cooling box filled with isopropanol. After the entire device is frozen solid, the handle is pulled and the frozen MN array is released from the mold and is ready for use.

[0186] Hold the handle and press the frozen MN array into the skin. The MN array penetrates the skin tissue to a depth of about 50-850 μm. Keep the handle against the skin for about 2 minutes to allow the ice to fully melt. Remove the handle, leaving the MN tip and its payload embedded in the skin tissue.

[0187] 4.3 Example 3

[0188] MN arrays (1×10) were fabricated according to the process described in Example 1. The three-electrode sensor was attached to the substrate layer via a soluble adhesive layer. The MN array was then brought into contact with the conjunctival meniscus in the patient's eye to extract a tear sample. The device was then immersed in a substrate solution (i.e., 5 mM K3[Fe(CN)6] / K4[Fe(CN)6] in 0.1 M KCl / PBS buffer) containing a biomarker recovery buffer containing 0.1% Triton-100 to recover IFN-γ from MNs. MNs will detach from the sensor surface, which allows for more complete IFN-γ recovery. After 10 minutes of recovery, the sensor was connected to an electrochemical device to detect electrical signals.

[0189] 5. How to use

[0190] In embodiments herein, the MNs and / or MN arrays herein may be part of or included in a monitoring system, such as for monitoring a biomarker. In one embodiment herein, the biomarker is IFN-γ found in tears. IFN-γ is a biomarker that may indicate ocular inflammation or the presence of an inflammatory eye disease.

[0191] Fig.10 A schematic diagram showing an embodiment of a method for using a MN patch according to the present invention is shown. Fig.10 , an eye 60 is shown containing tear fluid 62. A monitoring system 64 contains a microneedle patch 10 operably associated with an electrochemical sensor 66. When the MN patch 10 comes into contact with the tear fluid 62, the MN patch 10 extracts a sample of the tear fluid 62, which is then provided to the electrochemical sensor 66 for analysis.

[0192] Fig.11 A schematic diagram of an embodiment of a monitoring system 64 according to the present invention is shown. In this case, the monitoring system 64 is a tear biomarker detection system, wherein step 1 shows the MN array 10 on the horizontal substrate layer 20 extracting biomarkers 74 from tears from the eye 60.

[0193] Step 2 shows a reaction vessel 76 , such as a test tube, containing a release solution 78 , such as a biomarker recovery buffer, which causes the MN array 10 to release any absorbed biomarkers 74 .

[0194] Step 3 shows the MN array 10 releasing the biomarker 74 into the release solution 78. Step 4 shows the MN array 10 connected to the electrochemical sensor 80, which detects the biomarker 74 from the MN array 10 and / or the release of the biomarker 74 from the MN array 10 into the release solution 78.

[0195] In another embodiment herein, the biomarker is matrix metalloproteinase-9 (MMP-9) from tears, as it is considered a possible biomarker for a variety of conditions such as, for example, inflammation, dry eye, cancer.

[0196] In embodiments herein, anisotropic porous MNs, MN arrays, monitoring systems, etc. as described herein; or unloaded anisotropic porous MNs, unloaded MN arrays, unloaded monitoring systems, etc., can be used to collect samples, such as, for example, body fluids, liquids, and combinations thereof; or blood, urine, saliva, vaginal fluids, tears, and combinations thereof; or tears. Without wishing to be limited by theory, it is believed that the tip surface of the anisotropic porous MNs herein is very small and the cross-linked polymer matrix is ​​very soft, thus avoiding irritation to, for example, the surface of the eye. Therefore, using the anisotropic porous MNs herein to directly capture microliters of, for example, tear samples from the eye can reduce potential pain and irritation to the subject.

[0197] In embodiments herein, anisotropic porous MNs, MN arrays, monitoring systems, etc. as described herein; or unloaded anisotropic porous MNs, unloaded MN arrays, unloaded monitoring systems, etc. can be used to deliver a payload as described herein to a target. The target can be, for example, a specific part of a human or animal, such as, for example, the dermis, eyes, and / or other locations.

[0198] In embodiments herein, the anisotropic porous MN is kept in a mold until just prior to use, so as to protect the structure of the anisotropic porous MN from damage, contamination, etc., during, for example, storage, transportation, etc.

[0199] 6. Proof of Concept

[0200] Microneedle (MN) technology is rapidly expanding in research and clinical translation. It is a potent and non-invasive delivery platform for a wide range of drugs, sensors, and target tissues. Their integration with advanced materials, electronics, and various control systems has led to many applications in drug delivery, biosensing, and smart therapy systems.

[0201] Throughout the history of MN development, precise control of its microstructure is very important for designers. This precise control determines the ultimate performance of the MN device, including mechanical properties and the efficiency of drug delivery and body fluid sampling. Porous MNs have interconnected nano- or micron-sized pores through which fluids can be effectively transported via capillary action (because of the channel-like connected pores), thereby transporting the collected fluid to the analysis system or delivering the drug to the tissue. Compared with non-polymer porous MNs, polymer porous MNs are generally biocompatible and biodegradable, and can be easily manufactured without the need for complex micromachining processes and equipment and the requirement for a clean room environment.

[0202] It is noted that existing porous MNs in the prior art only have randomly interconnected pores without precise control over the pore diameter and running direction. In addition, due to the non-perforated structure, these porous MNs cannot be used to deliver cell-based therapeutics.

[0203] Reference now Fig.12 The inventors have discovered that incorporating anisotropic porous structures similar to xylem vessels 1210 in the MN device 12 enables efficient liquid transport and delivery of drugs of varying sizes from small molecules to cells, from the tip to the base.

[0204] Currently, in the prior art, porous structures are made by techniques such as emulsification post-bonding, hot embossing, and thermogenic leaching. However, none of these techniques can achieve the goals of the present invention. Ice templating 1220, also known as freeze casting, is a technique that exploits the highly anisotropic solidification behavior of a solvent (usually water) in a precursor solution, followed by freeze drying, to allow controlled molding of oriented porous materials. This technique is widely used in the biomedical field due to its precisely controllable pore size, good shape recovery, and irreversible compressive mechanical properties.

[0205] The resulting MN 12 with anisotropic pores (APC-MN) manufactured by ice molding 1220 is lightweight, flexible, and soft like a bird feather. It shows good liquid absorption capacity compared to isotropic porous polymer MNs. The study also showed that the pore size can be precisely controlled by adjusting the polymer concentration and freezing temperature. As the polymer concentration increases, the pore size decreases, while higher freezing temperatures lead to an increase in pore size. This unidirectional porous structure and feather-like texture make APC-MN 12 suitable for extracting tears 1230. Compared with commercially available tear test strips, APC-MNs have a smaller contact area with the ocular surface 1231, and the results of this study indicate that APC-MN 12 does not pose a risk to the ocular surface 1231. When used in rats with dry eye and diabetes, tear biomarkers were successfully recovered from APC-MNs, where the levels of biomarkers analyzed were consistent with those obtained from the test strips.

[0206] Further, MNs have also been used for cell delivery 1240. Briefly, due to capillary forces, a γδ T cell suspension was directly absorbed into APC-MNs 12. The cell-loaded APC-MNs were frozen to obtain sufficient mechanical strength for skin penetration. The γδ T cell-containing devices were then used to treat pleural mesothelioma and subcutaneous melanoma solid tumor mouse models.

[0207] 6.1 Fabrication and characterization of APC-MNs

[0208] Low temperature cross-linking is a process under semi-frozen conditions that results in a cross-linked polymer network around ice crystals. Subsequent thawing / removal of the ice crystals leaves behind a polymer material with an interconnected network surrounded by high density polymer walls. When a unidirectional freezing method is used in the process, the porous network will be oriented. If no temperature gradient is applied, the pores will be disordered.

[0209] Fig.13 a shows an embodiment of the fabrication process of APC-MN 12. Low temperature cross-linking is applied together with unidirectional freezing on a PDMS MN mold 30 containing a prepolymer solution 40. This solution 40 consists of a polymer (e.g. gelatin), EDC and NHS and is loaded into the tip hole of the PDMS mold. The base cavity of the mold is filled with pure water.

[0210] After covering the mold 30 with a tin foil patch that served as the base layer of the APC-MN 12, the sealed mold 30 was placed with the tin foil down on a cooling bath 50, where ice crystal growth was oriented from the base of the mold. This cross-linking at -20 degrees lasted for two days, and unreacted molecules were removed by thawing before freeze-drying to obtain APC-MNs.

[0211] Three other types of MNs were also manufactured, including isotropic porous cryogels (IPCs) that served as controls, freeze-dried hydrogels (LHs) and solid hydrogels (SHs). The manufacturing method of IPC-MNs is similar to that of APC-MNs, except that they are frozen in a uniform temperature environment, rather than frozen under a temperature gradient. Compared with the manufacturing process of APC-MNs and IPC-MNs (which involves freezing, cryogelation, thawing and freeze-drying), LH-MNs and SH-MNs are prepared by the order of cross-linking, freezing and freeze-drying or drying at room temperature. The main difference is the order of cross-linking and ice crystal growth. When ice crystal growth occurs first (APC-MNs and IPC-MNs), it causes larger ice crystal growth, thereby producing an interconnected porous structure. On the other hand, when cross-linked before freezing, it limits the growth of ice crystals, thereby producing relatively isolated pores.

[0212] like Fig.13 As shown in FIG. 1 , APC-MN 1201 is identical to SH-MN 2601 from a top view. However, a cross-sectional view of the APC-MN tip 1202 shows the porous structure of the lining. LH-MN 2501 has random and interconnected pores, while IPC-MN 2401 has random pores across the device. Fig.14a As shown in the SEM images of , these phenomena (different internal structures) are also seen when the mold changes from MN morphology to cylindrical shape. Fig.13d, The porous structures of APC-MNs, IPC-MNs, and LH-MNs were further stained with Cy3, and longitudinal sections of these MNs were imaged by fluorescence microscopy after cryosectioning.

[0213] The pore size of APC-MNs can be controlled by adjusting the freezing temperature and polymer concentration. Fig.14b The SEM images in Figure 2 show APC-MNs with polymer concentrations of 2.5 wt.%, 5 wt.%, 7.5 wt.%, and 10 wt.% fabricated using freezing temperatures of -20°C, -80°C, and -196°C. It was found that the pore size decreased with increasing polymer concentration but increased with increasing freezing temperature. In conclusion, anisotropic porous structures of MNs can be fabricated by a unidirectional freezing method.

[0214] 6.2 APC-MN for liquid extraction

[0215] 6.2.1 Liquid extraction capacity

[0216] Different APC-MNs with different pore sizes were prepared for liquid extraction by adjusting the polymer concentration (2.5wt.%, 5wt.%, and 7.5wt.%). The obtained APC-MNs were named APC2.5-MN, APC5-MN, and APC7.5-MN. The liquid extraction ability of APC-MNs was tested by absorbing a drop of Cy3 dye solution. As shown in Figure 14, regardless of the pore size, all these APC-MNs can absorb the liquid from the tip to the base within 1.5s. Compared with IPC-MNs, LH-MNs, and SH-MNs, at a specific polymer concentration (7.5wt.%), APC-MNs showed the fastest liquid absorption rate.

[0217] APC-MNs showed good liquid absorption properties due to the capillary forces formed by the aligned channels. Although the fabrication process of APC-MNs and IPC-MNs was similar, the liquid absorption rate of APC-MNs was faster than that of IPC-MNs due to the anisotropic porous structure. LH-MNs have relatively isolated pores, while the liquid flow in SH-MNs depends on hydrogel swelling, resulting in a much slower fluid absorption rate than that of APC-MNs.

[0218] 6.2.2 APC-MN for tear extraction

[0219] The excellent liquid absorption properties make APC-MNs a good candidate for tear extraction. Fig.15a shows an embodiment of a method of use 1500 according to the present invention, wherein the health of the eye 60 is monitored by using APC-MNs, the method of use comprising tear extraction, biomarker recovery and biomarker post-analysis.

[0220] To evaluate the risk assessment of APC-MNs for use in the eye, mechanical testing and mouse corneal layer damage testing were performed.

[0221] Fig.15 15b and 15c show the mechanical properties of APC-MN and SH-MN under shear force. APC-MN showed significantly weaker mechanical properties than SH-MN. After applying shear force, both APC-MN and SH-MN tips bent, but the shear force required for APC-MN bending was smaller, indicating that APC-MN is soft and friendly to eye application.

[0222] Furthermore, to ensure that APC-MNs did not damage the corneal layer of the eye, APC-MNs were administered to mouse eyes, and SH-MNs were used as a control group. Fig.15 c shows H&E staining of the corneal layers of untreated, APC-MN-treated and SH-MN-treated mice. No obvious damage was shown on the corneal layers treated with APC-MN, which was similar to the untreated group. For the SH-MN-treated group, obvious tip holes 1510 could be observed on the corneal layers. These data indicate that APC-MNs exhibit weaker mechanical properties than SH-MNs and do not harm the eyes.

[0223] Biomarker recovery tests were performed in vitro to verify whether the biomarkers could be released from the MN tips. FITC-dextran (10 kDa) was used as a model biomarker to simulate the recovery of tear biomarkers from APC-MNs fabricated with different polymer concentrations and different freezing temperatures. Fig.15 As shown in d, the FITC-dextran recovery rate of APC-MNs increased with decreasing freezing temperature or polymer concentration. APC5-MNs fabricated with a freezing temperature of -20 °C were selected as tear extraction devices because the biomarker recovery rate of APC5-MNs was comparable to that of the control group and had a better anisotropic porous structure than APC2.5-MNs.

[0224] In addition, if Fig.15As shown in e and 15f, real disease biomarkers in tears (glucose as a biomarker for diabetes and IFN-γ as a biomarker for dry eye disease) were used for release from APC-MNs, tear test strips (Surgi Edge, India), rayon swabs (Huayue, China), and cotton swabs (Wastons). The glucose recovery rate of APC-MNs was close to 100%, indicating that tear glucose can be completely released from APC-MNs. The percentage of IFN-γ recovered from APC-MNs was the highest compared to commercial tear test strips and swabs, indicating that APC-MNs have the potential to monitor eye health.

[0225] 6.3 APC-MNs for drug delivery

[0226] 6.3.1 Loading of drugs into APC-MNs

[0227] Different APC-MNs were prepared using gelatin concentrations of 2.5 wt%, 5 wt% and 7.5 wt%, and isotropic porous MNs were prepared using a gelatin concentration of 5 wt%. They were named APC2.5, APC5, APC7.5 and IPC5, respectively.

[0228] Fig.16 a shows a schematic diagram of an embodiment of a method 1600 for manufacturing APC-CryoMN. Freeze-dried hydrogel (gelatin concentration of 5 wt %, FH5) was also used to prepare a coated PDMS mold to compare the effects of FH5 and APC-MN on liquid absorption capacity. By using this ready-to-use mold 30 filled with APC-MNs, only 10 μL of the required liquid 54 is required to fill the tip, followed by adding a replenishing solution as the base, thereby greatly reducing the amount of liquid required for the MN mold 30. After placing the bracket or handle 58 on the mold and cooling them down, the APC-cryoMN12 can be stored for a long time at low temperatures (such as liquid nitrogen). Well-formed APC-cryoMN12 is formed after demolding and is ready for use. Therefore, the process of preparing cryoMNs using APC-MNs is relatively simple and efficient because no centrifugation or vacuuming is required.

[0229] To characterize the hydrophilic properties of APC-MNs, a drop of water was added to the PDMS negative molds including the unfilled (untreated) mold, the unfilled but hydrophilically treated (plasma-treated) mold, and the mold filled with APC-MNs (filled with APC-MNs) to measure the water contact angle.

[0230] Fig.16b shows an optical image indicating the water contact angles on an unfilled mold, an unfilled but hydrophilically treated mold, and a mold filled with APC-MNs. After 1.5 s, the water contact angle of the unfilled PDMS mold was 96.4°±1.6°, while the water contact angle of the hydrophilically treated mold was about 58.2°±7.1°. On the other hand, Fig.16 As shown in Fig. 3, the water contact angle of the mold filled with APC-MN was 27.2°±5.0° within 1.5 s and continued to decrease. The comparison of the water contact angles of the unfilled, unfilled but hydrophilic treated PDMS molds and the APC-MN filled PDMS molds is shown in Fig. Fig.16 h. The large water contact angle indicates the hydrophobic nature of the unfilled mold, while the hydrophilic treated mold shows improved hydrophilic properties. In contrast to the unfilled mold and the hydrophilic treated mold, the mold filled with APC-MNs shows hydrophilic properties. The liquid distribution in the tip holes of the mold filled with APCMNs was visualized using a Cy3 fluorescent dye solution.

[0231] like Fig.16 As shown in Figure d, compared with the unfilled group and the hydrophilic-treated group, the fluorescence of the group filled with APC-MNs filled the entire tip. The penetration distance of the dye in the unfilled mold and the hydrophilic-treated mold was 223.4±50.9μm and 486.4±25.5μm, respectively, while the penetration distance in the mold filled with APC-MNs was 897.6±26.7μm. Fig.16 Only in the mold filled with APC-MNs did the liquid show the ability to completely fill the tip holes of the PDMS mold, indicating that the oriented porous gelatin APC-MNs have excellent liquid absorption capacity.

[0232] It is an important feature that various therapeutic agents can be absorbed into the needle tip cavity of APC-MNs. Cy3 and FITC-dextran with different molecular weights (10 kDa and 150 kDa) (FD1W and FD15W) were used to simulate small molecules and various macromolecular drugs. In contrast to LH5-MNs, all APC2.5-MNs, APC5-MNs, APC7.5-MNs, and IPC5-MNs can absorb liquid into the tip cavity regardless of molecular weight. Fig.17aThe distribution of fluorescent dyes with different molecular weights (Cy3 and FITC-dextran with molecular weights of 10 kDa and 150 kDa or FD1W and FD15W) in the tips of APC2.5-MN, APC5-MN, IPC5-MN, LH5-MN, and APC7.5-MN is shown in Figure , where scale bar = 100 μm. Benefiting from the interconnected macroporous structure of APC-MN and IPC5-MN, liquid can penetrate and fill the entire tip pore regardless of pore size or orientation. However, small and isolated pores in the tip of the mold filled with FH-MN prevented the liquid from filling completely, resulting in the solution being trapped in the shallow layer. Lipid nanoparticles encapsulated with FITC (FITC-LNP) were also used to add to APC2.5, APC5, APC7.5, and IPC5-MN. Reference Fig.17b , the fluorescence of FITC-LNP was distributed in the tips of APC2.5-MN, APC5-MN, and APC7.5-MN, but very few FITC-LNP entered the tip of IPC5-MN.

[0233] 6.3.2 APC-MNs for cell delivery

[0234] Next, the construction of APC-cryoMNs was tested with γδ T cells. PBS with a mixture of 2.5% (v / v) DMSO and 100 mM sucrose was used as a cryoprotectant (CGM) to cryopreserve γδ T cells in cryoMNs.

[0235] To verify the feasibility of the fabrication process, CGM was first added to fill the tip of the APC-MN, followed by dripping the supporting solution to fill the base portion. After freezing for 24 h, the APC-cryoMN tip broke during the demolding process when the supporting solution was pure PBS buffer. However, when the base consisted of a polymer solution such as PBS with 10% HA and PBS with 10% PVA, the APC-cryoMN could be well demolded from the PDMS mold. Fig.18 Photographic images of demolded APC-cryoMNs are shown when using different base formulations (i.e., PBS, PBS with 10 wt.% 10 kDa HA, and PBS with 10 wt.% 13 kDa PVA). PBS with 10% PVA was ultimately chosen as the base solution because PVA is cheaper than HA and has been reported as a cryopreservative.

[0236] Reference again Fig.16f, When PBS with 2.5% (v / v) DMSO and 100 mM sucrose was used to fill the needle tip and PBS with 10% PVA was used as the MN base, the MN tip was well formed. The use of a 3D printed scaffold embedded in a supporting solution prevented the APC-cryoMN from being melted by the heat from the fingers. After melting in PBS, the CGM at the tip and base dissolved immediately, and the MN tip with the aligned porous structure detached from the scaffold. Close-up image of the APC-CryoMN after melting in PBS Fig.16 As shown in g, it can be seen that the microneedle 12 has a directional porous structure.

[0237] 6.3.3 Characterization of APC-cryoMNs

[0238] Since the target site of administration is human skin, the ice crystals will melt at room temperature (RT) and come into contact with the skin. Evaluation of the thawing temperature of APC-cryoMNs is very important to help us understand the appropriate time to use APC-cryoMNs. Fig. 20 As shown in a, after the APC-cryoMN was removed from the liquid nitrogen storage environment (-196°C), frost formed on the tip immediately and gradually increased at RT of 21°C, and a phase transition from ice to water occurred after 1 minute.

[0239] Thermal imaging was applied to record the temperature changes of APC-CryoMN during the thawing process. Due to the limited temperature range detection of the device, temperature changes were recorded when the temperature was above -40 °C. Fig. 20 b and 20d, the color of the selected tip area (white box) changes from dark blue to blue, indicating that at RT ( Fig. 20 b) and finger skin temperature ( Fig. 20 d) The temperature increases during the thawing process.

[0240] Fig. 20 c and Fig. 20 e shows the quantified temperature, which was quantified for the tip change curve to analyze the temperature at RT ( Fig. 20 c) and skin temperature ( Fig. 20 e) Lifespan of APC-cryoMN. The tip temperature increased from -196°C to -40°C, -30°C, and -20°C within 13.2s, 29.2s, and 31.5s, respectively ( Fig. 20 c). After about 1 min, the temperature was close to 0°C. When touched with a finger, the temperature of the APC-cryoMN tip increased to about -40°C, -30°C, -20°C, and 0°C after about 10.9s, 20.6s, 23.5s, and 33.1s, respectively ( Fig. 20d). After 55.2 s, the temperature was close to 15°C. These findings suggest that the ideal residence time of APC-cryoMN at RT should be within 29 s.

[0241] This is consistent with previous studies that cryoMNs can penetrate porcine skin within 30 s without any significant difference in penetration depth. In addition, during the cell thawing process, 3.5% of ice crystals were formed in the range of -40°C to -30°C, while 75% of ice crystals were formed between -10°C and 10°C. In order to minimize damage to cells by ice crystals, APC-cryoMNs should be applied within 29 s after removal from liquid nitrogen.

[0242] To ensure the skin penetration ability of APC-cryoMN, mechanical properties were measured by compression testing and porcine skin insertion. Typically, the skin insertion process takes several seconds. Therefore, a compression speed of 0.5 mm / s was set for mechanical testing.

[0243] like Fig. 20 As shown in f, except for APC-cryoMN (with 7.5wt% gelatin cryogel), cryoMN and APC-cryoMN can withstand a compressive force of 5.8N, which has been reported as skin penetration force. As the polymer concentration increases, the compressive force that APC-cryoMN can withstand decreases, indicating that the mechanical properties are weaker when a higher concentration of polymer is used to make the mold filled with APC-MN. This may be because the pore size becomes smaller with increasing polymer concentration, thereby preventing ice crystals from growing during the freezing process. APC5-cryoMN was applied to porcine skin and as shown Fig. 20 As shown in g, the stratum corneum 2010 was significantly damaged compared with the untreated skin 2020, indicating that APC5-cryoMN has sufficient skin penetration ability.

[0244] 6.3.4 In vitro testing of cell-loaded APC-cryoMNs

[0245] Cell-loaded APC-cryoMNs were prepared by dropping a cell suspension onto a mold filled with APC-MNs and then performing gradient freezing. Fig.19 and Fig.21a, Six types of cells (hMSC, C2C12, B16, 3T3, dendritic cells and γδT cells) were loaded to evaluate the cell penetration ability of APC-MN (including APC2.5-, APC5- and APC7.5-MN). IPC5-MN was used as a control, in which case a few cells could enter the needle tip. In contrast, all six types of cells could easily enter the tip of APC-MN. Similar to the distribution of LNP, fewer cells penetrated into the tip of the mold filled with IPC5-MN, while cells could be absorbed into the needle tip cavities of all anisotropic porous structures, indicating that the unidirectional porous structure can improve cell loading performance. Fig.19 Cell penetration of hMSCs, DCs, 3T3, B16, and C2C12 in different PDMS molds loaded with APC-MNs was demonstrated.

[0246] In addition, for cells with larger sizes ranging from 15 μm to 30 μm (including hMSC, C2C12, 3T3 and B16), the mold filled with APC2.5-MN showed a deeper penetration distance compared to the mold filled with APC5-MN and the mold filled with APC7.5-MN. However, for dendritic cells (cell size: 10 μm to 15 μm) and γδT cells (cell size: 5 μm to 7 μm), 8 and 9 cells reached a greater absorption depth within the tip holes of the mold filled with APC5-MN compared to the other two molds filled with APC-MN. In the case of the mold filled with APC5-MN, the smaller the cell, the greater the penetration depth. These results indicate that the adjustable pore size of APC-MN allows adaptation to different types of cells. Based on these results, APC5 porous MNs were selected for loading γδT cells into cryoMNs.

[0247] Since the base part of APC-cryoMN is made of PVA solution, once the APC-cryoMN melts, the needle tip will be detached from the MN base.

[0248] like Fig.21 As shown in b, when the APC-cryoMN was taken out of liquid nitrogen and placed in pre-warmed PBS, the tip 2110 loaded with γδ T cells detached. The cell viability of γδ T cells cryopreserved with cryogenic medium (PBS with 2.5% (v / v) DMSO and 100 mM sucrose) was tested in cryotubes and APC-cryoMNs. Fig.21c and 21d. Dead cells stained with ethidium homodimer-1 can be detected by flow cytometry due to the emission of fluorescence, while the remaining cells do not show any signal. The cell viability of γδT cells in the vial tube and APC-cryoMN was 48.9% and 48.4%, respectively, indicating that the mold filled with APC-MNs did not have any negative impact on the percentage of live cells after cell thawing. In addition, to ensure cell functionality, such as recognition and killing of tumor cells after APC-cryoMN insertion, surface biomarkers of γδT cells were detected after cell thawing. 98.8±1.1% of γδT cells still maintained the expression of Vδ2-TCR (T cell receptor) after one month of cryopreservation, as shown in Fig.21 e The percentage of Vδ2+ cells was similar to that of primary isolated Vδ2+ cells, indicating that surface biomarkers would not be altered after cryopreservation in APC-MNs.

[0249] 6.3.5 In vivo mesothelioma ACT via APC-cryoMN delivery γδ T cells

[0250] Mesothelioma is a type of aggressive solid tumor that is primarily caused by exposure to asbestos fibers. Mesothelioma originates in the mesothelium, a thin layer of tissue that covers many internal organs, and across which the tumor can spread.

[0251] Due to the lack of accurate and reliable biomarkers for detection, mesothelioma is often diagnosed at an advanced stage with limited treatment options. With the advancement of CAR T therapy, mesothelioma remains difficult to treat due to the lack of specific antigens to serve as targets. γδT cells have been studied as potential therapeutic cells against mesothelioma because they have tumoricidal activity without the need to recognize specific tumor antigens. The cytotoxicity of γδT cells against mesothelioma cells has been previously reported, and the cells can induce apoptosis via the classical pathway and have efficacy in tumor xenograft mouse models.

[0252] Based on these findings, APC-cryoMNs were used to load γδ T cells for ACT delivery against intrapleural mesothelioma in vivo. Fig. 22 . APC-cryoMN loaded with γδ T cells was applied to the posterior skin of tumor-bearing mice by pressing the scaffold on the posterior skin of tumor-bearing mice. To ensure that the needle tip remained in the skin layer, the scaffold was removed after 1 min when the APC-cryoMN was completely melted. The needle tip hole was immediately visible after the scaffold was removed and disappeared after 12 min.

[0253] To test whether Vδ2 T cells can infiltrate into tumors, Vδ2 T cells were administered by iv and APC-cryoMN in MSTO-luc-bearing mice 3 days after tumor injection. A total of 1×107 Vδ2 T cells were used for iv injection. Based on the cell viability of cryopreserved γδT after thawing, 2×107 Vδ2 T cells were used for APC-cryoMN to maintain the number of injected live cells compared to iv. MSTO-luc mice were sacrificed on days 1, 2, 3, and 4 after transfer. Spleen, lungs, liver, and tumors were extracted to detect the presence of Vδ2 T cells by flow cytometry. In the iv and APC-cryoMN groups, CD3 was detected in the spleen and lungs on the first day after Vδ2 T cell injection. + Vδ2 + However, in both groups, the detection values ​​of Vδ2 T cells in the spleen tended to decrease from day 1 to day 4 after Vδ2 T cell injection. Similarly, when intravenous injection was used, the detection values ​​of Vδ2 T cells in the lung also decreased from day 1 to day 4.

[0254] In contrast, when APC-cryoMNs were used, the detection of Vδ2 T cells in the lungs initially increased from day 1 to day 3 and then decreased on day 4. In the liver, the detection of Vδ2 T cells remained low in the iv injection group throughout the 4-day period, whereas it increased on day 2 and decreased on day 3 when APC-cryoMNs were used. Importantly, in both groups, the detection of Vδ2 T cells in the tumors increased from day 1 to day 4 after Vδ2 T cell injection, as shown in Figure 5. Fig.19 shown.

[0255] Note that in Fig. 22 In the figure, the scale bar is 200 μm. Data represent the mean ± SEM from 3 independent experiments. Two-way ANOVA statistical test was used. *P<0.05, **P<0.01, ***P<0.001.

[0256] Next, we sought to compare the efficacy of Vδ2 T cells given via iv 2010 and APC-cryoMN 2020 delivery in mice with mesothelioma. Fig.24 As shown in the timeline of a, one or two doses of Vδ2 T cells were administered on day 3 and / or day 6. Evaluation of tumor luciferase activity revealed that a single dose of Vδ2 T cells administered via iv injection and APC-cryoMN resulted in a reduction in tumor growth of approximately 16.4% and approximately 14.7%, respectively. Fig.24 b and 24c. In addition, Fig.24As shown in d, administration of two doses of Vδ2 T cells via iv injection of 2010 and APC-cryoMN 2020 resulted in a reduction in tumor growth of approximately 58.6% and approximately 62%, respectively, compared to PBS control. The values ​​of luciferase activity detected in MSTO-luc mice in each treatment group during the experiment are shown in Fig.23 a, where each group consisted of three to four mice. Fig.23 b Graph showing the body weight changes of the MSTO-luc mouse group.

[0257] Based on these results, APC-cryoMN-delivered Vδ2 T cells had similar efficacy in targeting mesothelioma as iv-injected Vδ2 T cells, and mice survived slightly longer by two days, as shown in Figure 2. Fig.24 As shown in d.

[0258] This has important implications, as APC-cryoMN-delivered Vδ2 T cells can traverse the dermis into the bloodstream but still infiltrate into different organs and tumor masses with an efficiency comparable to that of direct iv delivery into the blood circulation. Due to the non-invasive nature of APC-cryoMN, it may become an important tool for the development of ACT in the future.

[0259] Note that Fig.24 Each treatment group contained 3 to 4 mice. Data represent mean ± SEM. For C, two-way ANOVA statistical test was used. ***P < 0.001.

[0260] 6.3.6 APC-cryoMN-delivered Vδ2 T cells for in vivo melanoma therapy

[0261] Based on the above findings, the effectiveness of Vδ2 T cells administered via APC-cryoMN in targeting melanoma tumors was further explored in another xenograft mouse model. Fig.25 A375 cells were injected subcutaneously (sc) for four days to form solid tumors. Vδ2 T cells were delivered by iv injection and APC-cryoMN on days 4, 6, and 8 after tumor injection, respectively.

[0262] Fig.25 b shows a photographic representation of tumor volume in mice receiving PBS as a control, Vδ2 T by iv and APC-cryoMN routes over a period of 11 days; and Fig.25 c shows a graph indicating the tumor volumes extracted from mice from each experimental group over time. Compared to PBS control, administration of Vδ2 T cells via iv injection and APC-cryoMN resulted in a reduction in tumor growth of approximately 50.2% and approximately 59.4%, respectively. Fig.25As shown in Figures 25d and 25e, no significant weight loss was observed in the mice during the entire experimental period.

[0263] about Fig.25 , scale bar is 200 μm, and data represent mean ± SEM. For B, two-way ANOVA statistical test was used. ***P < 0.001.

[0264] 7. Further details

[0265] 7.1 Experimental Section

[0266] Fabrication of APC-MNs

[0267] The PDMSMN mold was prepared by applying a mixture of PDMS prepolymer and curing agent (mass ratio: 10:1) to a stainless steel MN patch (10×10) (Micropoint Technologies, Singapore) via casting. The mold was then degassed in a vacuum oven and cured at 70°C for 2 hours. Subsequently, the PDMS negative MN mold was obtained by demolding the mold from the stainless steel MN patch. The obtained PDMS mold was ultrasonically cleaned in 75% ethanol for 30 min and sterilized by ultraviolet exposure for 1 h, and then APC-MNs were filled inside.

[0268] To fill the MN cavity, a 50 μL volume of the prepolymer containing gelatin and EDC / NHS was first passed through a 0.22 μm syringe filter ( The filter unit (Merck, Merck) was sterilized and then cast into the PDMS mold, and then centrifuged at 4,000rpm for 3 minutes. After centrifugation, 40 μL of solution was removed, and 150 μL of deionized water was added to fill the base cavity of the mold, and then aluminum foil was covered on the top. The PDMS mold was placed on a cooling bath with aluminum foil facing down, and quickly cooled to -20°C from the bottom, while the top surface was kept at room temperature. The mold was then stored at -20°C for 48h to enable gelatin cryogel to be cross-linked. Afterwards, a dialysis process was performed overnight at room temperature using deionized water to remove any unreacted chemicals in the ice base and tip. The manufacturing process was carried out in a sterile environment of a biological safety cabinet (BSC, Thermo Fisher Scientific). Subsequently, the PDMS mold was freeze-dried at -80°C for 4h to obtain MNs filled with APC-MNs. The MNs filled with APC-MNs obtained were sealed in a sterile and dry environment for subsequent cell loading. Anisotropic porous cryogel MNs with different gelatin concentrations were fabricated and loaded into PDMS molds, the names of these cryogel MNs were the same as the samples in the previous section, namely APC2.5-MN, APC5-MN and APC7.5-MN. The preparation of the molds filled with IPC-MNs was similar to that of the molds filled with APC-MNs, but they were frozen in a -20 °C freezer without aluminum foil covering. To create the molds filled with LH-MNs, the negative MN molds were filled with the same gelatin prepolymer solution. After filling, the solution in the base cavity was removed, and the mold was frozen after the gelatin hydrogel in the needle tip was completely cross-linked at room temperature. Finally, the mold was freeze-dried.

[0269] 7.2 Hydrophilicity test

[0270] Water contact angle measurements were performed on the surfaces of the APC-MN filled molds by the sessile drop technique using an OCA15EC drop tensiometer (DataPhysics Instruments, Filderstadt, Germany). For contact angle measurements, a 10 μL drop of water was deposited on the surface and the drop profile was recorded to determine the contact angle at the water / solid / air interface.

[0271] 7.3 Cell culture

[0272] Human mesenchymal stem cells (hMSCs), mouse melanoma cell line (B16), mouse myoblast cell line (C2C12), mouse fibroblast cell line NIH / 3T3 (3T3), and human melanoma cell line (A375) were cultured in culture dishes with high glucose DMEM supplemented with 10% FBS and 1% P / S at 37°C and 5% CO2. Mouse bone marrow-derived dendritic cells (DCs) were cultured in complete RPMI 1640 medium supplemented with GM-CSF and IL-4 at 37°C and 5% CO2. Human γδ T cells and a human mesothelioma cell line expressing luciferase (MSTO-luc) were cultured in culture dishes with RPMI 1640 medium (ATCC modified) supplemented with 10% FBS and 1% P / S at 37°C and 5% CO2. The culture medium was changed every 2 or 3 days, and culture dishes with 80%-90% adherent cell confluence were used for further cell experiments.

[0273] 7.4 Drug and Cell Penetration Testing

[0274] Cy3 was used as a small molecule drug model to evaluate drug penetration ability. 10 μL of Cy3 solution was added to a single array mold filled with APC2.5-MN, a single array mold filled with APC5-MN, and a single array mold filled with APC7.5-MN (1×10). 10 kDa FITC-dextran and 150 kDa FITC-dextran were applied in the same manner as the macromolecular drug model. The mold filled with IPC-MN and the mold filled with LH-MN were compared with the mold of APC-MN. 10 μL of FITC-encapsulated lipid nanoparticles (FITC-LNP) were added to the mold filled with APC2.5-MN, the mold filled with APC5-MN, and the mold filled with APC7.5-MN, which was used as a matrix / formulation drug model. The mold filled with IPC-MN was compared with the mold filled with APC-MN. A cell density of 1×10 5 The cell penetration ability of the mold filled with APC-MNs was evaluated by six cell types with a cell / mL, including human mesenchymal stem cells (hMSCs), human γδT cells, mouse melanoma cell line (B16), mouse dendritic cells (DC), mouse myoblast cell line (C2C12), and mouse fibroblast cell line NIH / 3T3 (3T3). The distribution of Cy3, 10 kDa FITC-dextran, 150 kDa FITC-dextran, FITC-LNP, and six types of cells in the tip was observed by fluorescence microscopy (Ti2-A, Nikon).

[0275] 7.5 Preparation of APC-cryoMN make

[0276] APC-cryoMNs were prepared by adding 10 μL of PBS cryoculture medium with a mixture of 2.5% (v / v) DMSO and 100 mM sucrose directly to a mold filled with APC2.5-MNs, a mold filled with APC5-MNs, and a mold filled with APC7.5-MNs to fill the needle tip cavity. 120 μL of PBS, PBS with 10% (w / v) HA, or PBS with 10% (w / v) PVA was added to fill the base cavity. Afterwards, the samples were frozen in a cryogenic storage box frozen at -80°C for 24 h and finally stored in liquid nitrogen. The APC-MNs obtained were named APC2.5-cryoMN, APC5-cryoMN, and APC7.5-cryoMN, respectively.

[0277] 7.6 Mechanical testing of APC-cryoMNs

[0278] APC2.5-cryoMN, APC5-cryoMN and APC7.5-cryoMN were mechanically tested using an Instron tensile tester 5942. The APC-cryoMN used in this test was prepared by filling the base with a 10% (w / v) solution of PVA in PBS. After demolding, the APC-cryoMN was stored in liquid nitrogen until use. Two force load cells were pre-cooled with liquid nitrogen, and the APC-cryoMN was then placed with the tip facing up. The compression test was set to have a compression speed of 0.5 mm / s. An empty PDMS mold filled with PBS with 2.5% (v / v) DMSO and 100 mM sucrose at the tip and PBS with 10% (w / v) PVA at the base was used as a control group, i.e., cryoMN.

[0279] To verify the mechanical properties of skin penetration, APC-cryoMNs were further inserted into porcine cadaver skin obtained from a local supermarket. Porcine skin insertion was performed by applying thumb pressure. The penetrated porcine skin tissue was fixed with 4% paraformaldehyde and then stained with H&E for histological analysis according to the protocol provided by the manufacturer.

[0280] 7.7 Melting process

[0281] After APC-cryoMNs were taken out of liquid nitrogen, the thawing process of APC-cryoMNs was recorded by an infrared thermal imager (FLIR ONE) at room temperature and body temperature (finger skin).

[0282] 7.8 Purification and expansion of γδ T cells

[0283] Peripheral blood mononuclear cells (PBMC) were isolated from human blood obtained from the Hong Kong Red Cross using Lymphoprep density gradient medium. Tetra-pivaloyloxymethyl 2-(thiazol-2-ylamino)ethylidene-1,1-diphosphonate (PTA) was used to stimulate the expansion of PBMC in recombinant human IL-2 protein. PBMC was cultured with RPMI1640 culture medium and 10% FBS and incubated for a total of 13 days at 37 ° C, 5% CO2. During this 13-day period, 50% of the culture medium was replaced every 2-3 days. Then, γδT cells were purified using a human TCRγ / δ+T cell separation kit to obtain CD3+Vδ2+ cells with a purity of>95%.

[0284] 7.9 Fabrication of cell-loaded APC-cryoMNs

[0285] γδT cells were cultured at 5×10 8 The cells were suspended in a cryogenic medium (PBS supplemented with 2.5 v / v% DMSO and 100 mM sucrose) at a cell density of 10 cells / mL. 6 Cells) were added to the APC-MNs still inside the PDMS mold. 120 μL of 10% (w / v) PVA solution was then added to each mold to fill the base, and the pre-sterilized scaffold was placed on top. The molds were placed at -80°C for 24 h and then stored in liquid nitrogen. The APC-cryoMNs were demolded before use.

[0286] 7.10 Visualization of the tip loaded with γδ T cells after thawing of APC-cryoMN

[0287] After thawing, the detached APC-CryoMN tips loaded with cells were visualized. Specifically, the APC-CryoMN tips were first visualized by CellTracker according to the manufacturer's protocol. TM GreenCMFDA reagent was used to stain γδT cells. 10 μL of stained γδT cells were added at 5×10 8 The APC-cryoMNs were cryopreserved at a density of 10 cells / mL in a -80°C freezer for two days. Afterwards, the APC-cryoMNs loaded with cells were immediately placed in pre-warmed PBS. The tips loaded with fluorescent γδ T cells were then imaged by fluorescence microscopy (Ti2-A, Nikon).

[0288] 7.11 γδT cell viability assay try

[0289] The cell viability of γδ T cells in different cryoculture medium formulations was tested by staining dead T cell nuclei with propidium iodide (PI). 8γδT cells of 1 cell / mL were cryopreserved in cryotubes or APC-cryoMNs with PBS having 2.5% (v / v) DMSO and 100mM sucrose. Conventional cryoculture medium with 10% DMSO and 90% FBS was used as a control group to compare the cell viability in cryopreserved tubes. After being stored in liquid nitrogen for one month, the cells were quickly recovered in a preheated FBS-free culture medium containing 1 μM PI, and then incubated in the dark for 20 min at 37 ° C, 5% CO2. The cell suspension was washed with PBS buffer for 10 min using a centrifugal speed of 1200 rpm. The cell pellet was resuspended in 500 μL PBS buffer supplemented with 1% BSA. The stained dead cells were then detected by a BD Biosciences desktop flow cytometer and analyzed by FlowJo software (TreeStar, version 10.5.3).

[0290] 7.12 Changes in surface biomarkers of γδT after cryopreservation

[0291] The surface biomarker changes of γδT cells were detected by T cell receptor (TCR) Vδ2. After cryopreservation in PBS with 2.5% (v / v) DMSO and 100mM sucrose in liquid nitrogen for one month, the γδT cells were recovered in preheated culture medium and resuspended with PBS buffer supplemented with 1% BSA. TCRVδ2 monoclonal antibody was applied to the γδT cell suspension, followed by application of the secondary antibody goat anti-mouse 488 after washing. The γδT cells were then washed with PBS buffer and resuspended in 500 μL PBS buffer supplemented with 1% BSA. The immunostained Vδ2 cells were then measured by a BD Biosciences desktop flow cytometer, and the cells were analyzed by FlowJo software (TreeStar, version 10.5.3).

[0292] 7.13 Mesothelioma Xenograft Mouse Model

[0293] NOD.Cg-Prkdc scid Iq tm1Wjl / SzJ (NSG) mice were obtained from the City University of Hong Kong Laboratory Animal Research Unit. The construction of the luciferase reporter gene mesothelioma cell line was based on the reported work. 6 MSTO-luc was injected intrapleurally (i.pl) into NOD.Cg-Prkdc scid Iq tm1Wjl / SzJ (NSG) mice were used to establish pleural mesothelioma xenograft mice. The luciferase signal was monitored on the second day by NightOWL II LB 983 in vivo imaging system (Berthold Technologies). When the luciferase signal was detectable, the mesothelioma xenograft mouse model was ready for use. Four patches of APC-cryoMN loaded with Vδ2 T cells (5 × 10 per patch) were placed on 6 cells, a total of 2×10 7 cells) were applied directly to the skin of each mouse for one dose of treatment. After each dose, the mouse was treated with Tegaderm TM Film (3M) was used to cover the application site for protection. On the same day, 1×10 7 Vδ2 T cells were injected intravenously (iv) into another group of mice, and 100 μL of PBS was given to the control group.

[0294] 7.14 In vivo tracking of Vδ2 T cells delivered by APC-cryoMN

[0295] In order to track Vδ2 T cells in vivo, mice were killed 1-4 days after injecting a dose of Vδ2 T cells. Skin tissue was collected and embedded in low-temperature matrix embedding medium and frozen by liquid nitrogen for subsequent frozen section immunostaining analysis. Spleen, lung, tumor and liver tissues were collected and processed by grinding them through a 70 μm cell strainer after treating them with red blood cell lysis buffer. The cell suspension was then incubated in the dark for 30 min and then washed twice with FACS buffer. Afterwards, it was incubated at 4 ° C for 30 min in 100 μL FACS buffer (PBS containing 1% FBS) with conjugated antibodies CD3 and Vδ2 TCR for surface protein staining. After washing the cells with FACS buffer, the cells were then measured by flow cytometry (BD FACSymphony A1).

[0296] 7.15 Mesothelioma Treatment Using APC-cryoMNs Loaded with Vδ2 T Cells

[0297] 1-dose and 2-dose of APC-cryoMN loaded with Vδ2 T cells were administered to mesothelioma-bearing mice on day 3 and day 3 and day 6 after tumor injection, respectively. Vδ2 T cells were injected intravenously as the iv group, while PBS was used as the control group. The mesothelioma luciferase activity of APC-cryoMN (n=3 for 1-dose and 2-dose), iv (n=4 for 1-dose; n=3 for 2-dose) and control group (n=4) was measured by NightOWL II LB 983 in vivo imaging system (Berthold Technologies) before and after treatment on days 2, 5 and 9 after tumor injection. Mouse body weight was measured on days 0, 2, 5 and 9. The survival of mice was monitored on days 2, 5 and 9 after tumor injection until the end of the experiment.

[0298] 7.16 Melanoma treatment using APC-cryoMN loaded with Vδ2 T cells

[0299] For melanoma-bearing mice, 1×10 7 A375 cells were injected subcutaneously (sc) into the right hind flank of mice. Three doses of APC-cryoMN loaded with Vδ2 T cells (5×10 6 cells / patch, for one dose 2×10 7 4 patches of 10 cells / patch) were applied directly to the skin of each mouse and then treated with Tegaderm TM The membrane (3M) covered the application site for protection. The iv group (n=5) and the control group (n=4) were administered to compare with the APC-cryoMN group (n=4). The body weight of each mouse was monitored. The tumor length and tumor width of the mice were measured every 2-3 days until the end of the experiment. The formula Calculate tumor volume (mm 3 ). Mice were sacrificed on day 11 after tumor injection. Dissected tumors and skin were embedded in cryo-matrix embedding medium and frozen by liquid nitrogen for subsequent histological and immunostaining analysis.

[0300] 7.17 Histological analysis

[0301] Frozen tumor and skin tissues were cryosectioned into 10 μm thick slices by a cryostat (Cryostar NX70, ThermoFisher) and placed on adhesive microscope slides. The sliced ​​tissues were fixed and dehydrated and then stained with an H&E kit according to the provided protocol. The H&E stained tissue sections were imaged with a microscope (Ti2-A, Nikon).

[0302] For immunostaining, the thinly sliced ​​tissue was fixed with acetone and placed in antigen retrieval buffer at 95°C for 10 min. 10% normal goat serum was added to block for 30 min. The thinly sliced ​​tissue was stained with unconjugated primary TCR Vδ2 monoclonal antibody and then stained with conjugated secondary antibody. Mounting medium with DAPI was applied to stain the nuclei. The stained tissue sections were imaged with a confocal laser scanning microscope (SP8LIA++TRUE, LEICA).

[0303] 7.18 Statistical analysis

[0304] Statistical analysis was performed using one-way analysis of variance (ANOVA) to compare at least three groups, and unless otherwise specified, P values ​​and Student's t-test were used to obtain P value significance between two groups. P < 0.05 was considered statistically significant (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001). Unless otherwise specified, each experiment was repeated at least three times in triplicate.

[0305] Thus, the exemplary embodiments are fully described. Although the description refers to specific embodiments, it should be clear to those skilled in the art that the present invention can be practiced by changes in these specific details. Therefore, the present invention should not be interpreted as being limited to the embodiments set forth herein.

[0306] Although embodiments have been illustrated and described in detail in the above description, these embodiments should be considered as illustrative rather than restrictive, and it should be understood that only exemplary embodiments are shown and described, and these exemplary embodiments do not limit the scope of the present invention in any way. It is understood that any feature described herein can be used together with any embodiment. The exemplary embodiments do not mutually exclude or exclude other embodiments not described herein. Therefore, the present invention also provides an embodiment including a combination of one or more exemplary embodiments in the above exemplary embodiments. Without departing from the spirit and scope of the present invention, the present invention as set forth herein can be modified and altered, and therefore, only such restrictions as indicated by the appended claims should be applied.

Claims

1. A microneedle comprising a base and a tip distal to the base, wherein the microneedle is formed of an anisotropic porous composition, the anisotropic porous composition comprising a plurality of channels extending through the microneedle in a substantially uniform direction from a surface of the base toward an outer surface defined by the tip, and wherein the plurality of channels are adapted to enable fluid to flow therein. 2 . The microneedle of claim 1 , wherein each of the plurality of channels has a substantially uniform channel diameter.

3. The microneedle according to claim 2, wherein the channel diameter is about 20 μm, or about 30 μm, or between 20-50 μm; or between 20-150 μm, or between 85-145 μm. The microneedle according to claim 1 , wherein the flow of the fluid in the plurality of channels is unidirectional.

5. The microneedle of claim 4, wherein the plurality of channels have a first physical property when the fluid flows into the microneedle and have a second physical property when the fluid flows out of the microneedle, and wherein the first physical property is different from the second physical property.

6. The microneedle of claim 5, wherein the first physical property and the second physical property include morphological properties, mechanical properties, pore structure, and Young's modulus. The microneedle according to claim 1 , wherein the anisotropic porous composition is formed of a cross-linked polymer matrix.

8. The microneedle of claim 7, wherein the cross-linked polymer matrix comprises a plurality of monomers selected from the group consisting of gelatin, alginate, polyvinyl alcohol (PVA), poly(2-hydroxyethyl methacrylate) (PHEMA), polyacrylamide (PAAm), vinyl-modified hyaluronic acid (HA), methacrylated hyaluronic acid (MeHA), and combinations thereof. 9 . A microneedle patch comprising a substrate and at least one microneedle according to claim 1 disposed on the substrate.

10. The microneedle patch of claim 9, wherein the substrate is integrally formed with the microneedles, and wherein the plurality of channels extend through both the substrate and the microneedles from an outer surface of the substrate toward an outer surface defined by the tips of the microneedles.

11. The microneedle patch of claim 9, wherein the substrate is formed separately from the microneedles and is attached to the bases of the microneedles. The microneedle patch according to claim 11 , wherein the substrate is a metal sheet.

13. The microneedle patch of claim 11, wherein the substrate is a handle.

14. The microneedle patch of claim 9, further comprising an electrochemical sensor attached to the substrate.

15. The microneedle patch of claim 9, further comprising a payload preloaded within the microneedle patch.

16. The microneedle patch of claim 15, wherein the payload is selected from the group consisting of cells, drugs, extracellular vesicles, macromolecules, and combinations thereof.

17. The microneedle patch of claim 16, wherein the cells are selected from the group consisting of immune cells, antigen cells, stem cells, fibroblasts, melanocytes, hair follicle cells, β cells, therapeutic cells, preventive cells, and combinations thereof.

18. The microneedle patch of claim 16, wherein the extracellular vesicles are selected from the group consisting of exosomes, microvesicles, apoptotic bodies, autophagic extracellular vesicles, matrix vesicles, stressed extracellular vesicles, and combinations thereof.

19. The microneedle patch of claim 16, wherein the macromolecule is selected from the group consisting of genetic material, polypeptide, protein, deoxyribonucleic acid sequence (DNA), ribonucleic acid sequence (RNA), enzyme, antibody, and combinations thereof.

20. The microneedle patch according to claim 9, comprising a plurality of microneedles, wherein the plurality of microneedles form a microneedle array disposed on the substrate.

21. A method for manufacturing a microneedle, comprising the following steps: (i) casting a prepolymer solution comprising a plurality of monomers into a mold defined by at least one recess shaped in a microneedle structure, wherein the microneedle structure comprises a base and a tip distal to the base; (ii) freezing the prepolymer solution with a temperature gradient across the mold; (iii) cross-linking the plurality of monomers to form a cross-linked polymer matrix; (iv) freeze-drying the cross-linked polymer matrix to form an anisotropic porous composition defined by the microneedle structure, wherein the anisotropic porous composition comprises a plurality of channels extending through the anisotropic porous composition in a substantially uniform direction from a base surface toward an outer surface defined by the tip; and (v) removing the anisotropic porous composition from the mold.

22. The method according to claim 21, wherein the temperature gradient is provided by arranging the first surface of the mold at room temperature, or at a temperature between 4 and 28°C, or at a temperature between 18 and 24°C, and A second surface of the mold opposite to the first surface is further arranged at a temperature between -0°C and -300°C, or between -25°C and -285°C, or between -50°C and -250°C.

23. The method of claim 22, wherein the second surface of the mold is placed in contact with a cooling element selected from the group consisting of: liquid nitrogen, liquid helium, dry ice, and mixtures thereof.

24. A method according to claim 23, wherein the cooling element is in the form of a cooling bath.

25. The method of claim 21, wherein the cross-linking of the plurality of monomers is achieved by freezing the prepolymer solution at a sub-zero temperature.

26. The method of claim 21, wherein the cross-linking of the plurality of monomers is achieved by exposing the prepolymer solution to a UV source.

27. The method of claim 21, further comprising the step of thawing the cross-linked polymer matrix at room temperature prior to the lyophilization step (iv).

28. The method of claim 27, wherein the cross-linked polymer matrix is ​​thawed in deionized water for 1 hour to 3 days, or 4 hours to 2 days, or 6 hours to 36 hours.

29. The method of claim 21, wherein the mold is formed of a mold material selected from a first group consisting of: polymers, metals, and combinations thereof; or selected from a second group consisting of: polydimethylsiloxane (PDMS), steel, resins, and combinations thereof.

30. The method of claim 21, wherein each channel of the plurality of channels has a substantially uniform channel diameter.

31. The method of claim 30, further comprising the step of adjusting the channel diameter by varying the polymer concentration of the prepolymer solution.

32. The method of claim 30, further comprising the step of adjusting the channel diameter by varying the temperature gradient.

33. The method of claim 30, further comprising the step of adjusting the channel diameter to about 20 μm, or about 30 μm, or between 20-50 μm, or between 20-150 μm, or between 85-145 μm.

34. The method of claim 21, wherein the plurality of monomers are selected from the group consisting of gelatin, alginate, polyvinyl alcohol (PVA), poly(2-hydroxyethyl methacrylate) (PHEMA), polyacrylamide (PAAm), vinyl-modified hyaluronic acid (HA), methacrylated hyaluronic acid (MeHA), and combinations thereof.

35. A method of manufacturing a microneedle patch, comprising the steps of the microneedle manufacturing method according to claim 21, and further comprising the step of forming a substrate having at least one microneedle disposed thereon.

36. The method of claim 35, wherein the substrate is formed by casting the prepolymer solution into the mold beyond filling recesses of the mold along the mold walls.

37. The method of claim 35, wherein the substrate is formed by attaching a separate layer to the base of the microneedles.

38. The method of claim 37, wherein the separate layer is a metal sheet.

39. The method of claim 37, wherein the separate layer is a handle.

40. The method of claim 35, further comprising the step of loading a payload into the microneedle patch.

41. The method of claim 40, wherein the payload is selected from the group consisting of: a cell, a drug, an extracellular vesicle, a macromolecule, and combinations thereof.

42. The method of claim 41, wherein the cell is selected from the group consisting of immune cells, antigenic cells, stem cells, fibroblasts, melanocytes, hair follicle cells, beta cells, therapeutic cells, preventive cells, and combinations thereof.

43. The microneedle patch of claim 41, wherein the extracellular vesicles are selected from the group consisting of exosomes, microvesicles, apoptotic bodies, autophagic extracellular vesicles, matrix vesicles, stressed extracellular vesicles, and combinations thereof.

44. The method of claim 41, wherein the macromolecule is selected from the group consisting of genetic material, polypeptides, proteins, deoxyribonucleic acid sequences (DNA), ribonucleic acid sequences (RNA), enzymes, antibodies, and combinations thereof.

45. The method of claim 40, wherein the payload is loaded into the prepolymer solution when the polymer solution is cast in the mold.

46. ​​The method of claim 40, wherein the payload is loaded into the anisotropic porous composition prior to removing the anisotropic porous composition from the mold.

47. The method of claim 35, further comprising the step of attaching an electrochemical sensor to the substrate.

48. A method of using the microneedle patch according to claim 9, comprising the step of applying the microneedle patch to an application area to deliver a payload from the microneedle patch to the application area.

49. The method of use according to claim 48, wherein the application area is the skin surface.

50. The method of use according to claim 48, wherein the application area is the ocular surface.

51. The method of use of claim 48, wherein the application area is an oral cavity surface.

52. A method for using the microneedle patch according to claim 9, comprising the step of bringing a sample into contact with the microneedle patch to collect the sample.

53. The method of use according to claim 52, wherein the sample is tear fluid.

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