Tip-loaded microneedle array for percutaneous insertion

Abstract

To provide a method of forming a microneedle array that can include forming a microneedle array having one or more bioactive components.SOLUTION: The microneedle array can include a base portion and a plurality of microneedles extending from the base portion, and the one or more bioactive components are present in a higher concentration in the plurality of microneedles than in the base portion. The systems and methods disclosed herein include cutaneous delivery platforms based on dissolvable microneedle arrays that can provide efficient, precise and reproducible delivery of biologically active molecules to human skin. The microneedle array delivery platforms can be used to deliver a broad range of bioactive components to a patient.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Related applications This application claims the benefit of U.S. Provisional Patent Application No. 61 / 641,209, filed on 1 May 2012, which is incorporated herein by reference in its entirety.

[0002] field This disclosure relates to systems and methods for transdermal drug delivery, and more particularly to systems and methods for preparing and using soluble microneedle arrays.

[0003] Approval of government support This invention was made with government support under authorization numbers EB012776, AI076060, and CA121973, granted by the National Institutes of Health. The government has certain rights in this invention. [Background technology]

[0004] background The skin's prominent physical barrier function presents significant challenges for transdermal drug delivery. To address these challenges, various microneedle array-based drug delivery devices have been developed. For example, one conventional method uses solid or hollow microneedle arrays that contain no active ingredients. Such microneedle arrays can pre-treat the skin by perforating the stratum corneum and upper layers of the epidermis to enhance transdermal drug penetration before topical application of biopharmaceutical carriers or traditional patches. While this method has been shown to significantly increase skin permeability, it offers limited ability to control the dosage and amount of the drug or vaccine being delivered.

[0005] Another conventional method uses solid microneedles coated with the drug. While this method offers somewhat better dose control, it severely limits the amount of drug delivered. This drawback limits the widespread application of this technique, for example, by hindering the simultaneous delivery of optimal amounts of antigen and / or adjuvants in vaccine administration.

[0006] Another conventional method involves using hollow microneedles attached to a reservoir of the biopharmaceutical. The syringe-needle-like characteristics of these arrays can significantly increase the speed and accuracy of delivery, as well as the amount of cargo delivered. However, complex fabrication procedures and specific application settings limit the applicability of such reservoir-based microneedle arrays.

[0007] Another conventional method involves using biodegradable and soluble solid microneedle arrays. Current fabrication techniques for soluble polymer-based microneedles generally utilize a microcasting process. However, such conventional processes are wasteful because they are limited to the active ingredients that can be embedded in the array and require the active ingredients to be homogeneously embedded in the microneedles and their supporting structures. [Overview of the Initiative] [Problems that the invention aims to solve]

[0008] Therefore, while transdermal delivery of biologics using microneedle array-based devices offers attractive theoretical advantages over prevalent oral and needle-based drug delivery methods, significant practical limitations exist in the design and fabrication associated with microneedle arrays constructed using conventional processes. [Means for solving the problem]

[0009] Abstract The systems and methods disclosed herein include a skin delivery platform based on a soluble microneedle array that can result in efficient, precise, and reproducible delivery of bioactive molecules to human skin. The microneedle array delivery platform can be used to deliver a wide range of bioactive ingredients to patients.

[0010] The aforementioned and other purposes, features, and advantages of the disclosed embodiments will become more apparent from the following detailed description, which proceeds with reference to the accompanying drawings. In embodiments of the present invention, for example, the following items are provided. (Item 1) A dissolvable microneedle array for percutaneous insertion into a patient, One or more types of bioactive ingredients, Base, and Multiple microneedles extending from the base Includes, A soluble microneedle array in which one or more of the aforementioned biologically active components are present at a higher concentration within the plurality of microneedles than within the base. (Item 2) The microneedle array according to item 1, wherein substantially all of the one or more of the aforementioned bioactive components are located within the plurality of microneedles such that the base is substantially formed without containing any of the bioactive components. (Item 3) The microneedle array according to item 2, wherein the one or more types of biologically active components are locally concentrated within the plurality of microneedles such that they are generally present only in the upper half of each microneedle of the microneedle array. (Item 4) The microneedle array according to item 2, wherein the plurality of microneedles are pre-formed to have a shape including a first cross-sectional dimension at the top, a second cross-sectional dimension at the bottom, and a third cross-sectional dimension in the middle, the middle being located between the top and bottom, and the third cross-sectional dimension being larger than the first and second cross-sectional dimensions. (Item 5) The microneedle array according to item 4, wherein one or more of the aforementioned biologically active components are substantially concentrated in the region in the intermediate part or in the region above it. (Item 6) Each microneedle generally tapers to a point above the middle portion, and each microneedle generally tapers to a smaller cross-sectional dimension below the middle portion, the microneedle array according to item 5. (Item 7) Each microneedle contains a plurality of layers of a soluble biocompatible material, the microneedle array according to item 2. (Item 8) The soluble biocompatible material is carboxymethyl cellulose, the microneedle array according to item 7. (Item 9) The one or more bioactive components include at least two different bioactive components, the microneedle array according to item 2. (Item 10) The at least two different bioactive components are selected from the group consisting of chemotherapeutic agents, adjuvants, and chemoattractants for cancer chemoimmunotherapy applications, the microneedle array according to item 9. (Item 11) The bioactive components include an antigen and an adjuvant for vaccine application, the microneedle array according to item 9. (Item 12) The one or more bioactive components include at least one viral vector, the microneedle array according to item 2. (Item 13) The at least one viral vector includes an adenovirus vector, the microneedle array according to item 12. (Item 14) A method for fabricating a microneedle array, Applying a first solution of a soluble biocompatible material containing one or more bioactive components therein to a mold for manufacturing a microneedle array, Applying a second solution of a soluble biocompatible material containing no one or more active components to the mold for manufacturing the microneedle array, The process includes the step of drying the first and second solutions to form a solid microneedle array comprising a base and a plurality of microneedles extending from the base, A method wherein one or more of the aforementioned active ingredients are substantially concentrated within the plurality of microneedles. (Item 15) The method according to item 14, wherein the one or more of the bioactive ingredients are substantially concentrated in the upper half of each microneedle of the microneedle array. (Item 16) The method according to item 14, wherein the one or more biologically active components of the first solution include at least one viral vector. (Item 17) The method according to item 16, wherein the at least one viral vector comprises an adeno vector. (Item 18) A method for forming a microneedle array, A step of forming a sheet of material having multiple layers, wherein at least one of the multiple layers contains at least one bioactive component, The process includes the step of removing portions from a sheet of material until a microneedle array is formed having a base and a plurality of microneedles extending from the base, A method for removing portions from the sheet array, comprising forming the microneedle array such that the one or more bioactive components are concentrated within each of the microneedles of the microneedle array, and the one or more bioactive components are substantially absent from the base. (Item 19) The method according to item 18, wherein the method for forming a sheet of the material comprises spatially distributing the at least one bioactive component throughout the entire sheet of the material. (Item 20) The method according to item 19, wherein the spatially distributed bioactive component is covered with a subsequent layer of soluble biocompatible material before portions are removed from the sheet of material to form the microneedle array. (Item 21) The method according to item 18, wherein the at least one bioactive component of the first solution or suspension comprises at least one viral vector. (Item 22) The method according to item 21, wherein the at least one viral vector comprises an adeno vector. (Item 23) A method for forming a microneedle array, A step of forming a sheet of material having multiple layers, wherein at least one of the multiple layers contains at least one bioactive component, The steps include micromilling a sheet of the material to form a microneedle array, which includes a base and a plurality of microneedles extending from the base, A method wherein the at least one bioactive component is concentrated within each of the microneedles of the microneedle array, and the at least one bioactive component is substantially absent from the base. (Item 24) The act of forming a sheet of material having multiple layers, To create a base layer with substantially uniform thickness, a layer of carboxymethylcellulose hydrogel is prepared, The base layer is dried until it becomes substantially solid, The method involves providing one or more active layers on the base layer, wherein the one or more active layers have a substantially uniform thickness and contain a carboxymethylcellulose hydrogel and a bioactive component. The process involves drying the one or more active layers until they become substantially solid. The method described in item 23, including the method described in item 23. (Item 25) The act of micromilling the sheet of the aforementioned material, The method according to item 23, comprising micromilling a sheet of the material to form a plurality of microneedles having a substantially pyramidal shape. (Item 26) The act of micromilling the sheet of the aforementioned material, The method according to item 23, comprising forming the microneedle array such that each microneedle has a first cross-sectional dimension at the top, a second cross-sectional area at the bottom, and a third cross-sectional dimension in the middle, wherein the middle is located between the top and the bottom, the third cross-sectional dimension is larger than the first and second cross-sectional dimensions, and the at least one bioactive component is concentrated in or above the middle. (Item 27) The method according to item 23, wherein the at least one bioactive component comprises two or more different bioactive components. (Item 28) The method according to item 27, wherein the two or more different biologically active components are selected from the group consisting of chemotherapeutic agents, adjuvants, and chemoattractants for cancer chemoimmunotherapy. (Item 29) The method according to item 27, wherein the two or more different biologically active components include at least one antigen and at least one adjuvant for vaccine application. (Item 30) The method according to item 23, wherein the at least one biologically active component comprises at least one viral vector. (Item 31) The method according to item 30, wherein the at least one viral vector comprises an adeno vector. (Item 32) A device for delivering a microneedle array into target tissue, An applicator head sized to engage with the microneedle array for applying a force substantially perpendicular to the surface of the structural support of the microneedle array, power supply, An electromagnetic oscillator coupled to the aforementioned power supply, and Oscillator energy converter coupled to the electromagnetic oscillator Equipped with, The oscillator energy converter is configured to convert electromagnetic vibrations generated by the electromagnetic oscillator into mechanical motion of the applicator head. (Item 33) The apparatus according to item 32, wherein the applicator head is detachably coupled to the apparatus. (Item 34) The apparatus according to item 32, wherein the applicator head is made of a material that can withstand sterilization by autoclave. (Item 35) The apparatus according to item 33, wherein the power supply is detachably coupled to the apparatus. (Item 36) The apparatus according to item 33, wherein the electromagnetic oscillator is detachably coupled to the apparatus and is completely sealed to allow for fluid sterilization. (Item 37) The apparatus according to item 33, wherein the applicator head moves in a plane substantially perpendicular to the surface of the structural support of the microneedle array, and the movement of the applicator head in a plane parallel to the surface of the structural support of the microneedle array is substantially restricted. [Brief explanation of the drawing]

[0011] [Figure 1] Figure 1 shows exemplary microneedles and their dimensions. [Figure 2] Figure 2 shows an exemplary microneedle array and its dimensions. [Figure 3] Figures 3A and 3B show exemplary microneedles with the active ingredient loaded at their tips. [Figure 4] Figures 4A and 4B show exemplary microneedles with the active ingredient loaded at their tips. [Figure 5]Figures 5A and 5B show exemplary microneedles with the active ingredient loaded at their tips. [Figure 6] Figures 6A and 6B show exemplary microneedles with the active ingredient loaded at their tips. [Figure 7] Figure 7 shows a miniature precision micro-milling system used to manufacture microneedle master molds. [Figure 8] Figure 8 is an SEM image of a micromilled master mold with a pyramidal needle. [Figure 9] Figure 9 is an SEM image of a pyramid production mold. [Figure 10] Figure 10 is an SEM image of a magnified segment of a manufacturing mold, showing a pyramidal needle molding well in the center of the image. [Figure 11] Figures 11A to 11D show exemplary CMC solids and embedded active ingredients. [Figure 12] Figures 12A and 12B show exemplary CMC solids and embedded active ingredients. [Figure 13] Figure 13 is a schematic diagram of an exemplary vertical multilayered structure and a method for fabricating it. [Figure 14] Figure 14 is a schematic diagram of an exemplary microneedle array fabricated using stratification and spatial distribution techniques for embedded active ingredients. [Figure 15] Figure 15 is a schematic diagram of an exemplary microneedle array fabricated in a spatially controlled manner. [Figure 16A] Figure 16A shows SEM images of multiple pyramidal molded microneedles. [Figure 16B] Figure 16B is an SEM image of a single pyramidal molded microneedle. [Figure 17] Figure 17 is an SEM image of a columnar molded microneedle. [Figure 18] Figure 18 is a micrograph of a pyramidal-shaped microneedle. [Figure 19] Figure 19 is a micrograph of a columnar molded microneedle. [Figure 20] Figure 20 shows various microneedle geometries that can be formed using a micro-milled master mold or by directly micro-milling a block of material. [Figure 21] Figure 21 shows a test apparatus for conducting destructive and piercing tests. [Figure 22] Figure 22 shows the force-displacement curves for a columnar microneedle (left) and a pyramidal microneedle (right). [Figure 23] Figure 23 shows finite element models of the deflection of columnar microneedles (left) and pyramidal microneedles (right). [Figure 24] Figure 24 shows various stereomicrographs of pyramidal (A, C, E) and columnar (B, D, F) microneedle penetrations in skin explants. [Figure 25] Figures 25A, 25B, and 25C illustrate the effectiveness of microneedle arrays in penetrating skin explants. [Figure 26] Figures 26A and 26B show in vivo delivery of granular material to lymph nodes in the cutaneous inflow area of ​​microneedle array-immunized mice. [Figure 27] Figure 27 is a bar graph showing the immunogenicity of model antigens delivered by microneedles. [Figure 28] Figure 28 is a bar graph showing the stability of the active cargo of the CMC microneedle array during storage. [Figure 29] Figures 29A and 29B show the induction of apoptosis in epidermal cells delivered by a microneedle array using Cytoxan® (cyclophosphamide). [Figure 30]Figure 30 shows the geometric shapes of microneedles that can be formed by directly micro-milling a block of material. [Figure 31] Figure 31 is a stereomicroscope image of a directly fabricated solid CMC microneedle array. [Figure 32] Figure 32 is a stereomicroscope image of a portion of the microneedle array shown in Figure 31. [Figure 33] Figure 33 is a schematic cross-sectional view of a mold assembly for directly creating blocks or sheets of material for micromilling. [Figure 34] Figure 34 is a schematic cross-sectional view of a drying apparatus that can be used to dry blocks or sheets of material for direct micromilling. [Figure 35] Figure 35 shows the flow cytometry analysis of GFP-expressing 293T cells. [Figure 36] Figure 36 shows the stability of viruses embedded in microneedles after several days of storage. [Figure 37] Figure 37 shows the expression and immunogenicity of adeno vectors delivered by a microneedle array. [Figure 38] Figure 38 shows an applicator for inserting a microneedle into target tissue. [Figure 39] Figure 39 shows the design of an applicator head for use with the applicator shown in Figure 38. [Figure 40] Figure 40 is a schematic diagram of the dimensional movement of the applicator head. [Modes for carrying out the invention]

[0012] Detailed explanation The following descriptions are illustrative in nature and are not intended to limit the scope, applicability, or configuration of the disclosed embodiments. Various modifications to the described embodiments can be made in the function and arrangement of the elements described herein without departing from the scope of this disclosure.

[0013] As used in this application and in the claims, the singular forms "a," "an," and "the" include the plural form unless otherwise clearly required by context. Furthermore, the term "includes" means "comprises." In this specification, the terms "biologics," "active ingredients," "bioactive components," "bioactive materials," or "cargo" refer to pharmaceutical active agents, such as analgesics, anesthetics, antiasthmatics, antibiotics, antidepressants, antidiabetics, antifungals, antihypertensives, anti-inflammatorys, antineoplastics, anxiolytics, enzymatically active substances, nucleic acid constructs, immunostimulants, immunosuppressants, vaccines, etc. Bioactive materials may include soluble materials, insoluble but dispersible materials, natural or formulated macro, micro, and nano-particulate matter, and / or mixtures of two or more soluble materials, dispersible insoluble materials, and natural and / or formulated macro, micro, and nano-particulate matter.

[0014] In this specification, the term “pre-formed” means that a structure or element is manufactured, constructed and / or formed into a particular shape or configuration before use. Thus, the shape or configuration of a pre-formed microneedle array is the shape or configuration of that microneedle array before one or more of its microneedles are inserted into a patient.

[0015] While the operations of exemplary embodiments of the disclosed methods may be described in a specific sequential order for the sake of convenient presentation, it should be understood that the disclosed embodiments may encompass orders of operations other than the specific sequential order disclosed. For example, sequentially described operations may, in some cases, be rearranged or performed simultaneously. Furthermore, descriptions and disclosures made in relation to a particular embodiment are not limited to that embodiment and may apply to any disclosed embodiment.

[0016] Furthermore, for the sake of brevity, the accompanying drawings may not show the various configurations (easily identifiable to those skilled in the art based on this disclosure) in which the disclosed systems, methods, and apparatus can be used in combination with other systems, methods, and apparatus. Additionally, this description occasionally uses terms such as “generate” and “provide” to describe the disclosed methods. These terms are high-level abstractions of the actual operations that can be performed. The actual operations corresponding to these terms may be modified according to a particular implementation and are easily identifiable to those skilled in the art based on this disclosure.

[0017] Tip-loaded microneedle array Dissolvable microneedle arrays enable efficient and safe delivery of drugs and vaccines to skin and mucous membrane surfaces. However, the homogeneous nature of conventional microneedle array fabrication can lead to inefficient drug delivery. While the drug or other cargo to be delivered to the patient is generally incorporated throughout the microneedle array matrix, in reality, only the microneedles enter the skin, and therefore only the cargo contained within the volume of individual needles is deliverable. Consequently, the majority of the drug or other cargo localized in non-needle components (e.g., the array's supporting structure) is never delivered to the patient and is generally discarded as waste.

[0018] Figures 1 and 2 show exemplary dimensions of microneedles and microneedle arrays. Based on the exemplary sizes shown in Figures 1 and 2, a microneedle array containing an active ingredient uniformly distributed throughout the array will exhibit more than 40 percent active ingredient waste. For example, an array with a total area of ​​61 mm² 2 The area of ​​the microneedle array is 36 mm². 2 In this case, the utilization rate of the active ingredient is less than 60 percent. The dimensions reflected in Figures 1 and 2 show specific sizes and shapes of microneedles, but it should be understood that similar waste exists in any other size of microneedle array where the active ingredient is homogeneously distributed throughout the array, regardless of the size of the array or the shape of the microneedles involved.

[0019] The systems and methods described herein provide novel microneedle array fabrication techniques that utilize fully soluble microneedle array substrates and unique microneedle geometries, enabling effective delivery of a wide range of active ingredients, including a broad range of protein and / or small molecule pharmaceuticals and vaccines.

[0020] As described in detail herein, in some embodiments, the technology can also uniquely enable the simultaneous co-delivery of multiple chemically distinct active ingredients for multifunctional drug delivery. Examples of the usefulness of these devices include, for example, (1) simultaneous delivery of multiple antigens and adjuvants to induce a multivalent immune response related to infection prevention and cancer therapy, (2) co-delivery of chemotherapeutic agents, immunostimulants, adjuvants and antigens to enable simultaneous adjuvant oncology therapy, and (3) topical skin delivery of multiple therapeutic agents without systemic exposure to treat a wide variety of skin diseases.

[0021] In some embodiments, the systems and methods disclosed herein relate to novel fabrication techniques that enable the incorporation of various active ingredients into the needle tips. Thus, by localizing the active ingredients in this manner, the remainder of the microneedle array volume can be prepared using less expensive matrix materials that are inactive and generally considered safe. The final result is a greatly improved efficiency of drug delivery, based on (1) a reduction in waste of undeliverable active ingredients incorporated into the non-needle portions of the microneedle array, and (2) a higher drug concentration in the needle tips penetrating the skin. This technological advance dramatically improves economic feasibility proportional to the cost of drug cargo and increases the effective cargo delivery capacity per needle of these novel microneedle arrays.

[0022] Figures 3A, 3B, 4A, and 4B illustrate various embodiments of microneedle arrays in which the active ingredient is concentrated at the tip of each microneedle in the array. Thus, in contrast to conventional microneedle arrays, the active ingredient is not present at a uniform concentration throughout the microneedle array, as there is little to no active ingredient in the supporting base structure. Furthermore, in some embodiments (for example, as shown in Figures 3A, 3B, 4A, and 4B), not only is there little to no active ingredient in the supporting structure, but the location of the active ingredient is concentrated in the upper half of each individual microneedle in the array.

[0023] Figures 5A and 5B show exemplary images of microneedles in a microneedle array containing active ingredients concentrated in the upper half of each individual microneedle. The active ingredients are shown as fluorescent particles concentrated at the tips of the microneedles, which are defined by the extent of the microneedle extending from the base in a narrowing and / or tapering manner. The base further extends from the supporting structure of the array.

[0024] Figures 6A and 6B show additional exemplary images of microneedles in a microneedle array containing the active ingredient concentrated in the upper half of each individual microneedle. In Figure 6A, the active ingredient, BSA-FITC, is concentrated at the tip of the microneedle. In Figure 6B, the active ingredient, OVA-FITC, is also concentrated at the tip of the microneedle.

[0025] As described above, in some embodiments, individual microneedles may contain the active ingredient only in the upper half of the microneedle. In other embodiments, individual microneedles may contain the active ingredient only in the tip or the narrowed portion near the tip of the microneedle. In yet another embodiment, individual needles may contain the active ingredient throughout the entire microneedle portion extending from the support structure.

[0026] The following embodiments describe various exemplary methods for fabricating microneedle arrays containing one or more active ingredients concentrated in the upper half and / or tip of each microneedle in the respective microneedle array.

[0027] Microneedle arrays fabricated by sequential microforming and spin drying methods. The following steps describe an exemplary method for fabricating a microneedle array using sequential microforming and spin drying. The active ingredient / cargo can be prepared in a suitable solvent at the desired useful concentration. As described herein, the solvent for the active ingredient(s) may be cargo-specific and may include a wide range of liquids, including, for example, water, organic polar and / or nonpolar liquids. Examples of active ingredients are discussed in more detail below, as is various information about these active ingredients, including the tested maximum loading capacity of various microneedle arrays.

[0028] If necessary, and required for specific applications, multiple loading cycles can be performed to achieve higher active cargo loading. Furthermore, multiple active cargoes can be loaded as a composite solution in a single loading cycle or as a single solution over multiple cycles (e.g., by repeating the loading cycles described below), depending on the specific cargo compatibility requirements of each cargo. In addition, granular cargoes (including those with nano- and micro-sized geometries) can be prepared as suspensions at the desired particle number / volume density. [Examples]

[0029] (Example 1) a) In the micromilling embodiment, as described in more detail below, the working stock solution / suspension of the active cargo is, for example, on a surface area of ​​1 cm². 2 Approximately 40 μl per unit can be applied to the surface of a mold for manufacturing microneedle arrays.

[0030] b) To fill the microneedle array manufacturing mold with needles in the working cargo stock, the microneedle array manufacturing mold, including the active cargo(s), can be centrifuged at 4500 rpm for 10 minutes.

[0031] c) Excess cargo solution / suspending can be removed from the surface of the mold for manufacturing microneedle arrays, mold surface area 1 cm² 2 Each can be washed with 100 μl of phosphate-buffered saline (PBS) or the solvent used to prepare the working stock of the activated cargo.

[0032] d) A mold for manufacturing a microneedle array containing an active cargo stock solution / suspension in the needle cavity can be spin-dried at the desired temperature for 30 minutes at 3500 rpm while continuously flowing a purge gas through a centrifuge at 0-50 L / min to facilitate the concentration of the drying active cargo(s) at the needle tip. The purge gas can be introduced into the centrifuge chamber through a tubular inlet. The moisture content can be reduced using a dehumidifier regulated to the desired temperature and recirculated into the centrifuge chamber. The purge gas can be air, nitrogen, carbon dioxide, or another inert or active gas, depending on the requirements of the specific cargo(s). The flow rate is measured by a flow meter and controlled by a circulation pump device.

[0033] e) Mold area 1 cm² for manufacturing microneedle arrays 2 100 μl of CMC90 hydrogel at 20% H2O can be added to the surface of a mold for manufacturing microneedle arrays and loaded into the structural components of a microneedle array device.

[0034] f) The mold for manufacturing the microneedle array can be centrifuged at the required temperature at 4500 rpm for 10 minutes in the centrifuge chamber without purge gas exchange, thereby filling the needle cavities of the microneedle array mold with CMC90 hydrogel. A 30-minute incubation period can then be followed to allow rehydration of the previously deposited active cargo(s) at the tips of the microneedle array.

[0035] g) The mold for manufacturing the microneedle array can be centrifuged at the required temperature at 3500 rpm for 3 hours or more while a constant purge gas of 0 to 50 L / min is flowed through the centrifuge chamber, thereby spin-drying the MNA device to a moisture content of less than 5%.

[0036] h) The dried microneedle array device can then be separated from the mold used to manufacture the microneedle array and stored under desired conditions. In some embodiments, the CMC90-based device may be storable between approximately 50°C and -86°C.

[0037] Examples of microneedle arrays carrying active cargo loaded at the fabricated tip can be seen in Figures 3A to 6B.

[0038] Micro-milled master molds and spin-molded microneedle arrays In the following embodiments, a micromilling step is performed to create microneedle arrays of various specifications. However, it should be understood that the following embodiments describe certain details of microneedle array fabrication that may be applicable to processes for fabricating microneedle arrays without a micromilling step, including the process described above in the previous embodiments.

[0039] The following embodiments describe apparatus and methods for fabricating soluble microneedle arrays using a master mold formed by a micromilling technique. For example, a microneedle array can be fabricated based on a master mold (positive) - manufacturing mold (negative) - array (positive) methodology. Micromilling techniques can be used to generate various microscale geometric shapes in substantially any type of material, including metal, polymer, and ceramic components. Micromilled master molds of various shapes and configurations can be effectively used to produce multiple identical female molds. The female molds can then be used to microcast various microneedle arrays.

[0040] Figure 7 shows an example of a precision micromilling system that can be used to fabricate a microneedle master mold. Mechanical micromilling uses microscale (e.g., as small as 10 μm) milling tools within a precision computer-controlled miniature machine tool platform. The system may include a microscope for viewing the surface of the workpiece being cut by the microtool. The microtool can be rotated at ultra-high speeds (200,000 rpm) to cut the workpiece and create the desired shape. As described above, the micromilling process can be used to create complex geometric features using many types of materials. For example, various types of shaping tools, including carbide microtools, can be used in the micromilling process. However, in a preferred embodiment, diamond tools can be used to fabricate a microneedle array on a master mold. Diamond shaping tools may be preferred over other types of shaping tools because they are harder than conventional materials such as carbides and can result in cleaner cuts on the surface of the workpiece.

[0041] The master mold can be micromilled from a variety of materials, including, for example, Cirlex® (DuPont, Kapton® polyimide), which is the master mold material described in the exemplary embodiment. The master mold can be used to produce flexible manufacturing molds from suitable materials, such as SYLGARD® 184 (Dow Corning), which is the production material described in the following exemplary embodiment. It is desirable that the master mold be made of a reusable material so that a single master mold can be repeatedly used to produce a large number of manufacturing molds. Similarly, it is desirable that each manufacturing mold can produce multiple microneedle arrays.

[0042] Master molds can be manufactured relatively quickly using micro-milling technology. For example, a master mold containing a 10mm x 10mm array with 100 microneedles can be micro-milled in less than two hours, and in some embodiments, less than 30 minutes. Therefore, the short ramp-up time makes it possible to rapidly manufacture different geometries, thereby enabling the rapid development of microneedle arrays and facilitating the experimentation and research of various microneedle parameters.

[0043] The master mold material is preferably able to be cleanly separated from the manufacturing mold material and preferably able to withstand any elevated curing temperature that may be required to cure the manufacturing mold material. For example, in the embodiment shown, the manufacturing mold material is a silicone compound SYLGARD® 184 (Dow Corning), which generally requires a curing temperature of about 80-90 degrees Celsius.

[0044] Master molds can be made in various sizes. For example, in an exemplary embodiment, the master mold was made from 1.8 mm thick Cirlex® (DuPont, Kapton® polyimide) and 5.0 mm thick acrylic sheets. Each sheet can be initially flattened with a micro-milling tool, raising the position where the microneedles are created from the rest of the surface. To create the microneedle features (e.g., defined by the master mold), the micro-tool can be used in conjunction with a numerically controlled micro-milling machine (Figure 1). In this manner, the micro-milling process can provide complete control over the dimensions, sharpness, and spatial distribution of the microneedles.

[0045] Figure 8 is a scanning electron microscope (SEM) image showing the structure of a micromilled master mold with multiple pyramidal needles. As shown in Figure 8, circular grooves can be formed around the microneedle array of the master mold to create an annular (e.g., circular) wall section within the manufacturing mold. The circular wall section of the manufacturing mold can facilitate the spin casting process discussed below. It should be understood that while the wall section shown in Figure 9 and the respective master mold structures shown in Figure 8 are circular, other geometric shapes of wall sections or housings can be obtained. For example, depending on which shape is desired for the microneedle array device, the housing can be formed in a variety of shapes, including, for example, squares, rectangles, trapezoids, polygons, or various irregular shapes.

[0046] As discussed above, the manufacturing molds can be made from SYLGARD® 184 (Dow Corning), a two-component transparent curable silicone elastomer in which SYLGARD® and a curing agent can be mixed in a ratio of 10:1. This mixture can be degassed for about 10 minutes, applied to the master mold to form a layer of approximately 8 mm, then degassed again for about 30 minutes, and cured at 85°C for 45 minutes. After cooling to room temperature, the master mold can be separated from the cured silicone, and the silicone manufacturing mold was trimmed to the edge of the circular wall section surrounding the array (Figure 9). A large number of manufacturing molds (e.g., 100 or more) can be produced from a single master mold, and any apparent degradation of the Cirlex® or acrylic master mold, if any, is very slight.

[0047] Figure 9 is an SEM image of the pyramid manufacturing mold created as described above. Figure 10 shows a magnified segment of the manufacturing mold with a pyramid needle forming well in the center of the image. The forming well is configured to receive a substrate (and any components added to the substrate) for forming microneedles having an outer shape defined by the forming well.

[0048] To construct a microneedle array, a substrate can be used to form portions of each microneedle containing a bioactive component and portions that do not. As discussed above, each microneedle may contain the bioactive component only in the microneedle, or in some embodiments only in the upper half of the microneedle, or in other embodiments only in the portion of the microneedle that tapers near the tip. Therefore, to control the delivery of the bioactive component(s) and control the cost of the microneedle array, it is preferable that each microneedle has a portion containing a bioactive component and a portion that does not contain a bioactive component. In the embodiments described herein, the portion that does not contain a bioactive component includes the support structure of the microneedle array and, in some embodiments, the base (e.g., the lower half) of each microneedle in the array.

[0049] Various materials can be used as substrates for microneedle arrays. The structural substrates for biodegradable solid microneedles most commonly contain poly(lactic acid-co-glycolic acid) (PLGA) or carboxymethylcellulose (CMC) based formulations, but other bases can also be used.

[0050] CMC is generally preferred over PLGA as a substrate for the microneedle arrays described herein. PLGA-based devices may be limited to drug delivery and vaccine applications due to the relatively high temperatures (e.g., above 135 degrees Celsius) and vacuum required for fabrication. In contrast, CMC-based matrices can be formed at room temperature by a simple spin-casting and drying process, making CMC microneedle arrays more desirable for incorporating sensitive biopharmaceuticals, peptides, proteins, nucleic acids, and various other bioactive components.

[0051] CMC hydrogels can be prepared from low-viscosity sodium salts of CMC with or without the active ingredient (as described below) in sterile dH2O. In exemplary embodiments, CMC can be mixed with sterile distilled water (dH2O) and the active ingredient to achieve a CMC concentration of about 25 wt%. The resulting mixture can be stirred until homogeneous and equilibrated at about 4°C for 24 hours. During this period, the CMC and any other components may be hydrated and a hydrogel may be formed. The hydrogel can be degassed under vacuum for about 1 hour and centrifuged at about 20,000 g for 1 hour to remove any remaining micro-sized bubbles that may interfere with the spin-casting / drying process of the CMC microneedle array. The dry matter content of the hydrogel can be tested by drying a fraction (10 g) of the hydrogel at 85°C for about 72 hours. Ready-to-use CMC hydrogels should preferably be stored at about 4°C until use.

[0052] The active ingredient can be incorporated into the CMC hydrogel at a relatively high (20-30%) CMC dry biopharmaceutical weight ratio before the spin-casting process. The array is spin-cast at room temperature, and the process can be made compatible with the functional stability of a wide range of bioactive ingredients. Since the master mold and manufacturing mold can be reused for numerous production cycles, production costs can be greatly reduced. The resulting dehydrated CMC microneedle array is generally stable at room temperature or slightly lower (e.g., around 4°C), preserving the activity of the incorporated biopharmaceutical and facilitating easy, low-cost storage and distribution.

[0053] In an exemplary embodiment, the surface of the manufacturing mold can be covered with approximately 50 μl of CMC hydrogel (for a mold with a diameter of 11 mm) and spin-cast by centrifugation at 2,500 g for approximately 5 minutes. After the initial CMC hydrogel layer, another 50 μl of CMC hydrogel can be layered onto the mold and centrifuged at 2,500 g for approximately 4 hours. At the end of the drying process, the CMC microneedle array can be separated from the mold, trimmed from excess material at the edges, collected, and stored at approximately 4 degrees Celsius. The manufacturing mold can be cleaned and reused for further casting of the microneedle array.

[0054] In some embodiments, the CMC solid can be formed with a layer that does not contain the active ingredient and a layer that contains the active ingredient. Figures 11A to 11D show CMC solids with different shapes (Figures 11A and 11B), as well as CMC solids with an embedded active cargo in the upper layer that becomes the portion of the microneedle containing the active ingredient after micromilling. Figure 11C shows micron-sized fluorescent particles layered on the surface of the inactive ingredient-containing layer, and Figure 11D shows an example of toluidine blue layered on the surface of the inactive ingredient-containing layer.

[0055] Figures 12A and 12B also show CMC solids with different shapes, with Figure 12B showing a square shape and Figure 12B showing a rectangular shape. Both CMC solids can be milled to dimensions for further processing as described herein. It should be understood that the geometry and active cargo shown herein are not intended to be limited to exemplary embodiments.

[0056] (Example 2) CMC solids can be prepared using a defined geometry and an active cargo content in one or more layers of the prepared structure. Examples of active cargo integrated into CMC solids are described in more detail herein. After constructing a CMC solid containing embedded active cargo contained in at least one layer of the CMC solid, the CMC solid can be milled to project-specific dimensions and micromilled to fabricate the microneedle devices described herein.

[0057] (Example 3) In another embodiment, one or more layers of the active cargo can be embedded in a CMC solid for direct micromilling of the microneedle array. Figure 13 shows representative samples of vertical multilayer deposition and CMC embedding of active cargo on a CMC solid for direct micromilling of MNA devices.

[0058] In one exemplary method, a microneedle array can be fabricated by preparing a CMC solid having a defined geometry and containing no active cargo. The blank CMC solid can then be milled to the desired dimensions.

[0059] As shown in Figure 13, the active cargo(s) can be deposited onto the CMC solid in a project-specific geometric pattern to specifically include the active cargo(s) at the tip of the micromilled MNA device.

[0060] For example, as a method for depositing active cargo onto a CMC solid blank, 1) Direct printing using micronozzle-assisted droplet deposition, 2) Movement from a pre-printed matrix, 3) Droplet deposition using a computer-controlled robotic system We can list some examples.

[0061] Figure 14 shows the stratification and spatial distribution of embedded active cargo within a CMC solid block. After the first layer is deposited (A), it can be covered with a CMC layer (B), which provides a surface for subsequent deposition of active cargo (C). This process can be repeated until all desired layers are deposited and enclosed within a solid CMC block suitable for the micromilling process (D-F).

[0062] Figure 15 shows a schematic cross-section of a CMC block enclosing the active cargo deposit in a spatially controlled manner (A). This method allows for three-dimensional control and placement of active components after micromilling in an MNA device (B). In panel (B) of Figure 15, the placement of the active cargo is shown along the stem of the active cargo, but by controlling the milling process, the placement can be controlled vertically from the tip of the microneedle to the base. The colors represent different active components or different amounts / concentrations of the same material.

[0063] Therefore, a method is provided for vertically layering and depositing active cargo onto microneedles by sequentially depositing one or more active cargoes on the surface of a CMC solid, either in contact with each other or separated by layers of CMC. In some embodiments, the cargo can be spatially separated by horizontal pattern deposition of the active cargo. By combining vertical and horizontal patterns of active cargo deposition, the three-dimensional delivery and distribution of each defined active ingredient can be achieved, and the waste of active ingredients during the fabrication of the microneedle array can be further reduced.

[0064] Microneedle-integrated adenovector The following embodiments relate to soluble microneedle arrays, such as those described herein, which incorporate infectious viral vectors into a soluble matrix of the microneedle array. Using this technique, for the first time, live viral vectors can be incorporated into microneedle arrays. As described herein, incorporating viral vectors into the disclosed microneedle arrays stabilizes the viral vectors, and as a result, they maintain their infectivity after incorporation and after long-term storage. When microneedle array-integrated adenovectors (MIAs) are applied to the skin, skin cells are transfected. In the context of vaccines, we have demonstrated that applying MIA encoding HIV antigens to the skin results in a potent HIV-specific immune response. These results are described in detail in the following examples.

[0065] (Example 4) The method for preparing microneedle-integrated adenovectors described herein preserves the viability of adenovirus particles during preparation and dry storage. These steps were specifically designed based on the physical and chemical properties of the CMC microneedle array. The viability of the virus in the CMC microneedle array is - By including low viscosity carboxymethylcellulose (CMC90) at a final concentration of 2.5% (Step 2), and - Timed and temperature-controlled spin-drying concentration of adenovirus particles at the tip of the microneedle array device (step 6), - This was achieved by controlled partial rehydration of the needle-tip loaded adenovirus particles (step 8).

[0066] Preparation of tip-loaded microneedle-integrated adenovectors (MIAs): 1) Trehalose storage buffer (5% trehalose Sigma-Aldrich USA, 20 mM Tris pH 7.8, 75 mM NaCl, 2 mM MgCl2, 0.025% Tween80) in 2 × 10 9Resuspend the adenovirus particles at a density of particles / ml. 2) Mix the resuspended virus stock with an equal volume of 5% CMC90 prepared in trehalose storage buffer to yield an adenovirus working stock at a density of 1×10 9 particles / ml. 3) Add the adenovirus working stock suspension to the surface of the mold for manufacturing the micro-needle array at 40 μl per 1 cm 2 surface area (as described in detail in other embodiments of this specification). 4) Centrifuge the mold at 4500 rpm for 10 minutes at 22 °C to fill the needle tips with the adenovirus working stock. 5) Remove the excess virus stock and wash the surface of the mold with 100 μl of phosphate-buffered saline (PBS) solution per 1 cm 2 surface area of the mold. 6) Partially spin-dry the micro-needle array mold containing only the adenovirus stock solution in the cavities of the needles at 3500 rpm for 10 minutes at 22 °C. 7) Add 100 μl of 20% structurally non-cargo-containing CMC90 hydrogel in H2O per 1 cm 2 surface area of the mold to the surface of the micro-needle array mold to form the structure of the MIA device. 8) Centrifuge at 4500 rpm for 10 minutes at 22 °C to fill the cavities of the needles with 20% CMC90 and incubate for 30 minutes to rehydrate the dried adenovirus particles inside the tips (Steps 3 - 6, above). 9) Spin-dry the MIA device to a moisture content of less than 5% at 3500 rpm for 3 hours at 22 °C while flowing a constant air of 10 L / min through the centrifuge chamber by centrifugation. 10) Demold the dried MIA device and store it at 4 °C or -80 °C.

[0067] (Example 5) <0000​​Final products containing virus particles / MNA were prepared. A control blank MNA was prepared in the exact same manner but without the virus. Batches of Ad5.EGFP MNA and control MNA were stored at RT, 4°C, and -86°C, and viral stability was evaluated by infection assays. The specific transduction activity of MNA-integrated Ad5.EGFP virus was evaluated in vitro using 293T cells. 2 × 10⁶ cells were placed in a 6-well plate. 6 Cells were seeded in one well and transduction was performed in two ways using diluted virus suspensions, suspension + empty MNA (control), or Ad5.EGFP MNA, stored at RT, 4°C, and -86°C for specified times. Untransduced wells were included as negative controls. First, the cell population was analyzed for GFP expression by flow cytometry after 24 hours (representative histograms are shown in Figure 35).

[0068] As shown in Figure 35, incorporating Ad5.EGFP into MNA does not reduce transduction efficiency. Flow cytometry analysis of GFP-expressing targeted 293T cells versus untransfected control cells 24 hours after transduction with the same titer of Ad5.EGFP in the suspension or incorporated in the CMC patch. Figure 36 shows the stability of MNA-embedded Ad5.EGFP virus. GFP gene expression was assayed by flow cytometry as shown in Figure 37 and normalized to the infection efficiency of Ad5.EGFP suspensions stored at -86°C.

[0069] The infection efficiency using the MNA Ad5.EGFP virus was found to be 87.92 ± 4.5%, which is similar to that observed with conventional -86°C stored Ad5.EGFP suspensions (Figures 35 and 36), suggesting that the manufacturing process does not adversely affect the transduction efficiency of Ad-EGFP virus particles. To evaluate infectivity over time, the transfection efficiency of freshly prepared -86°C stored Ad5.EGFP suspensions was compared to the efficiency of MNA-integrated Ad5.EGFP stored for extended periods at RT, 4°C, or -86°C. Infectivity over up to 365 days (normalized against Ad5.EGFP suspension + empty CMC patch) is reported (Figure 36). These results suggest that the infectivity of MNA Ad5.EGFP is remarkably stable under storage conditions of either 4°C or -86°C, and somewhat stable for up to 30 days at RT.

[0070] These results demonstrate that microneedle array-delivered Ad-transgenes are expressed in the skin and induce a potent cellular immune response. To specifically evaluate gene expression in vivo, the inventors determined GFP expression in the skin after conventional intradermal injection (ID) or microneedle array-mediated intradermal delivery. The inventors determined that GFP expression was locally delivered by ID injection or via single microneedle array application. 8Individual Ad5.GFP virus particles were delivered (Figure 37). Skin was collected after 48 hours, frozen-sectioned, counterstained with blue fluorescent DAPI to identify cell nuclei, and then imaged by fluorescence microscopy. Significant cellular GFP expression was observed after both ID and microneedle array delivery. To assess immunogenicity, we evaluated antigen-specific lytic activity in vivo after single ID or microneedle array immunization without boosting. For this purpose, we immunized groups of mice with an E1 / E3 deletion Ad5-based vector encoding either codon-optimized full-length SIVmac239 gag or SIVmac239 gag p17 antigen (Ad5.SIV gag, Ad5.SIV gag p17). An empty vector was used as a control (Ad5). The inventors observed potent and similar levels of in vivo lytic activity specific to the dominant SIV gag p17-derived peptide KSLYNTVCV (SIVmac239 gag 76-84) after ID or microneedle array immunization using Ad5.SIV gag or Ad5.SIV gag p17 (Figure 37, CTL).

[0071] The microneedle array technology disclosed herein can also facilitate clinical gene therapy. This addresses, for example, at least two major limitations of conventional methods. First, it enables the stabilization and storage of recombinant viral vectors over extended periods. By making live viral vectors resistant to high and low temperatures, along with proven serum equivalents for frozen liquid formulations, microneedle array stabilization alleviates the pressures associated with the "cold chain." Furthermore, integration into a microneedle array enables precise, consistent, and reproducible delivery of viral vectors that are not possible with conventional methods. Finally, the viral vectors are repackaged within a biocompatible, completely disposable microneedle array, which directs delivery precisely to the surface of the skin, requiring only one delivery device.

[0072] Such gene delivery platforms are useful in bringing patient-friendly clinical gene therapies. Because these microneedle arrays are engineered not to penetrate to the depths of vascular or nerve structures, gene delivery to human skin is both painless and non-invasive. Furthermore, the fabrication process is flexible, enabling simple, rapid, and low-cost production, along with the potential for efficient scale-up. Also, as the final MIA device, it is stable at room temperature and inexpensive to transport and store. Combined, these structural and manufacturing advantages enable broad, rapid clinical deployment, making this gene delivery technology readily applicable to the prevention and / or treatment of a wide range of human diseases. Moreover, this technique can be extended to other vector-based vaccine platforms (e.g., vaccinia virus, AAV, etc.) that are currently limited by the same constraints. For at least these reasons, the disclosed microneedle arrays and methods of using them represent a significant advancement in the field of recombinant gene therapy.

[0073] Microneedle array - Exemplary active ingredients Various active ingredients are described in detail below. For convenience, the following examples are based on a microneedle array of 6.3 × 6.3 mm. This size, and therefore cargo delivery, can be modified by increasing or decreasing it by 2 to 100 times.

[0074] Common considerations regarding the maximum active cargo volume include, for example, the total needle volume in the array and the solubility of the active component(s) in the solvent (generally expected to be less than 50%). [ka] [ka] [ka]

[0075] Lead loading of live adenovirus generally includes the following modifications: a) The hydrogel suspension for tip loading contains 5% trehalose and 2.5% CMC90. b) The process temperature is maintained at 22°C.

[0076] Furthermore, lentiviral vectors generally require processing at 4°C and vapor trap-based humidity control. Additionally, short epitope peptides are typically solubilized in DMSO, with a solvent evaporation time of 4 hours during tip loading.

[0077] Microneedle structure and shape It should be understood that in each of the following embodiments, one or more layers of the active ingredient may be provided within the microneedles of the microneedle array described above. Therefore, for example, in some embodiments, the active ingredient is provided only within the scope of the microneedles, not within the structural support of the array, as shown in Figure 15. Furthermore, in other embodiments, the active ingredient is concentrated in the upper half of the microneedle, as shown at the tip of the microneedle in Figures 3A to 4B.

[0078] Figures 16A and 16B are SEM images of a CMC microneedle array formed by multiple pyramidal projections (i.e., microneedles). The average tip diameter of the pyramidal needles shown in Figure 16A is approximately 5–10 μm. As shown in Figure 16B, the sides of the pyramidal needles can be formed with curved and / or arcuate surfaces that can facilitate insertion into the skin.

[0079] Figure 17 is another SEM image of a single needle in a microneedle array. The microneedle shown in Figure 17 is a base-extended columnar molded CMC microneedle. A base-extended columnar microneedle includes a base whose cross-section is generally polygonal (e.g., rectangular) and a projection extending from the base. The projection has a substantially rectangular lower section and a tip that generally tapers to a point. The tip is generally pyramidal in shape, and the exposed surface of the pyramid can be flat or arched. The projection can be more than half the total length of the needle.

[0080] Figures 18 and 19 show micrographs of pyramidal (Figure 18) and columnar (Figure 19) molded CMC microneedles. Because pyramidal needles have a continuously increasing cross-sectional profile (dimensions) from the needle tip to the needle base, the force required to keep the pyramidal needle in the skin increases as it enters the skin. In contrast, columnar needles generally have a continuous cross-sectional profile (dimensions) once they reach the generally rectangular portion of the projection. Therefore, columnar needles may be preferable to pyramidal needles because they can allow the needle to be introduced into the skin with less force.

[0081] Figure 20 shows schematic diagrams of microneedle shapes and structures generally suitable for fabrication by spin-casting material onto a master mold formed by micro-milling. The shapes and structures shown in Figure 20 do not contain any undercuts and therefore generally do not interfere with the molding / demolding process. The structures in Figure 20 include (a) nearly pyramidal microneedles, (b) "sharp" columnar microneedles (without the base member in Figure 8), (c) "broad" columnar microneedles, (d) "short" columnar microneedles (with a short columnar section and a longer pointed section), and (e) "filleted" columnar microneedles.

[0082] While the volume of pyramidal microneedles may be larger than that of columnar microneedles, their progressively increasing cross-sectional profiles (dimensions) require progressively increasing insertion force. Therefore, the geometry of pyramidal microneedles can reduce insertion depth and decrease effective delivery volume. Conversely, smaller cross-sectional areas and larger aspect ratios of columnar microneedles can lead to a lower fracture force limit. A smaller apex angle α results in a "sharper" microneedle tip. However, making the apex angle too small (e.g., less than approximately 30 degrees) can reduce the resulting microneedle volume and mechanical strength to undesirable levels.

[0083] The penetrating force of a microneedle is inversely proportional to its sharpness, which is characterized not only by the (apex) angle of the microneedle but also by the radius of the microneedle tip. The apex angle is determined by the master mold geometry, while the sharpness of the tip also depends on the reliability of the mold. Micro-rimming the master mold described herein makes it possible to increase the precision of the mold geometry, thereby increasing the precision and reliability of the resulting manufacturing mold and the microneedle array formed by the manufacturing mold.

[0084] As micromilling precision increases, it becomes possible to incorporate more precise and dense elements into mold designs. For example, as discussed in the following section, forming fillets in the base of columnar microneedles can significantly increase the structural integrity of the microneedles, thereby reducing the likelihood of them breaking or fracturing upon impact with the skin. These fillets can significantly increase the strength of the microneedles without interfering with their functional requirements (e.g., penetration depth and biopharmaceutical volume). Such fillets are very small features that can be difficult to create in master molds formed by conventional techniques. However, the micromilling techniques described above make it possible to include such small features with little to no difficulty.

[0085] Mechanical integrity and penetration capability The microneedle array is preferably configured to minimize pain and bleeding by penetrating the stratum corneum to deliver its cargo (e.g., a biopharmaceutical or bioactive ingredient) to the epidermis and / or dermis, while preventing penetration into deeper layers that may contain nerve endings and blood vessels. To evaluate the mechanical viability of the fabricated microneedle arrays, tests were performed on pyramidal and columnar microneedle arrays (e.g., shown in Figures 7B and 8) as representative variations of the array geometry. The first set of tests demonstrated the fracture limit of the microneedles, involving pushing the microneedle array against a solid acrylic surface at a constant approach speed while simultaneously measuring the force and displacement until fracture occurred. The second set of tests demonstrated the microneedle's ability to penetrate human skin explants.

[0086] Figure 21 shows the test apparatus designed for functional testing. The sample (i.e., microneedle array) was mounted on a fixture and advanced towards a fixed acrylic artifact (PMMA surface) at a constant speed of approximately 10 mm / second using a computer-controlled moving stage (ES14283-52 Aerotech, Inc.). A three-axis dynamometer (9256C1, Kistler, Inc.) receiving the acrylic artifact enabled highly sensitive force measurements.

[0087] Figure 22 shows the force-displacement curves of the data measured during fracture testing. The curve on the left represents the data obtained from testing columnar microneedle samples, and the curve on the right represents the data obtained from testing pyramidal microneedles. As seen in Figure 22, these two types of fracture of microneedles are remarkably different; pyramidal arrays undergo plastic deformation (bending), while columnar arrays exhibit fracture of the columns at their base. This different fracture behavior is associated with considerably different displacement-force data. The fracture (breakage) event can be easily identified from the displacement-force data, as shown in the figure. Based on the data obtained, the fracture point of columnar microneedles was found to be 100 mN on average. Since only about 40 mN of force is required to penetrate the stratum corneum, the microneedles are strong enough to penetrate human skin without fracture. Furthermore, since the similarity between the microneedle tips and the acrylic artifact cannot be fully established, the actual fracture limit is probably considerably higher than 100 mN (i.e., the microneedles fractured in a sequential manner rather than the simultaneous fracture of almost all of them).

[0088] The pyramidal microneedles exhibited a continuously increasing force signature without any apparent signs of fracture. To identify the fracture limit of the pyramidal microneedles, interruption tests were performed, in which the microneedles were advanced and retracted into the artifact by a specific amount and examined by optical microscopy. This process was continued until fracture was observed. For this purpose, fracture was defined as the bending of the pyramidal microneedles beyond 15 degrees.

[0089] To further analyze the fracture of the microneedles, a finite element model (FEM) of the microneedle array shown in Figure 23 was developed. A series of nanoindentation tests (using a Hysitron nanoindenter) were performed to obtain the mechanical properties (elastic modulus and strength limit) of the CMC material. The average elastic modulus and yield strength of the CMC material (as prepared) were 10.8 GPa and 173 MPa, respectively. This indicates that the prepared CMC material has a higher elastic modulus and yield strength than both PMMA (elastic modulus: 3.1 GPa, yield strength: 103 MPa) and polycarbonate (elastic modulus: 2.2 GPa, yield strength: 75 MPa), demonstrating superior strength and stiffness of the CMC material compared to other polymers.

[0090] Using this data, a series of FEM simulations were performed. The FEM model predicted that the fracture limits for pyramidal and sharp column (width = 134 μm) microneedles with a height of 600 μm, an apex angle of 30 degrees, and a fillet radius of 20 μm were 400 mN (pyramid) and 290 mN (sharp column) for asymmetric loading (5-degree loading orientation difference). Considering that the minimum piercing force requirement is approximately 40 mN, the pyramidal and sharp column microneedles should have safety factors of approximately 10 and 7.25, respectively.

[0091] When the fillet radius was doubled to 40 μm, the fracture load of the column increased to 350 mN, and when the fillet radius was reduced to 5 μm, the fracture load decreased to 160 mN. This is close to the fracture load determined experimentally. The height and width of the column had a significant effect on the fracture load. For example, for a column with a width of 100 μm, increasing the height from 500 μm to 1000 μm reduced the fracture load from 230 mN to 150 mN. For a column with a height of 750 μm, reducing the width to 75 μm resulted in a fracture load of 87 mN.

[0092] To evaluate penetration capabilities, pyramidal and sharp columnar microneedle arrays were tested for perforation into water-based model elastic substrates and full-thickness human skin. Figure 24 shows stereomicrographs of pyramidal (panels A, C, and E) and columnar microneedle arrays (B, D, and F) 4 minutes after exposure to the model elastic material. In particular, toluene blue tracer dye was deposited on the model elastic substrates (panels C and D) or freshly cut full-thickness human skin explants (panels E and F) after application of pyramidal or columnar microneedle arrays.

[0093] The model elastic substrate contained approximately 10% CMC and 10% porcine gelatin in PBS gelled at approximately 4°C for over 24 hours. Immediate contact between the needle tip and the water-based model elastic material was prevented by covering the surface of the elastic material with Parafilm approximately 100 μm thick. To enable stereomicroscopic imaging, trypan blue tracer dye (Sigma Chem., catalog number T6146) was incorporated into the CMC hydrogel at a concentration of 0.1%. Patches were applied using a spring-loaded applicator and analyzed after approximately 4 minutes of exposure. Based on the physical findings of the dye in the target substrate, the dissolution of two microneedles with different geometric shapes was remarkably different.

[0094] Sharp columnar needles applied to a model elastic substrate released substantially more tracer dye into the gel matrix than observed with the pyramidal design (Figure 24, C vs. D). Images of the recovered patches (Figure 24, A vs. B) were consistent with this finding, as the degradation of the sharp columnar needles was more advanced than that of the pyramidal needles. To extrapolate this analysis to a more clinically relevant model, pyramidal and columnar microneedle arrays were applied to freshly excised full-thickness human skin explants using the same force from a spring-loaded applicator. Consistent with the results from the elastic model, the pyramidal microneedle array deposited significantly less tracer dye than the sharp columnar microneedle array (Figure 24, E vs. F).

[0095] To further evaluate penetration and assess delivery efficacy to human skin, CMC microneedle arrays were fabricated using BioMag (Polysciences, Inc., catalog no. 84100) beads or fluorescent granular tracers (Fluoresbrite YG 1 μm, Polysciences Inc., catalog no. 15702). Pyramid CMC microneedle arrays containing fluorescent or solid granules were applied to living human skin explants as previously described. Five minutes after application, surface residue was removed, and the skin samples were frozen-sectioned and then counterstained with toluene blue for imaging by optical microscopy (Figures 25A and 25B) or fluorescence microscopy (Figure 25C).

[0096] The pyramidal CMC microneedles effectively penetrated the stratum corneum, epidermis, and dermis of living human skin explants, as evidenced by the deposition of Biomag beads lining the through-cavities corresponding to the insertion points of individual needles (representative sections shown in Figures 25A and 25B). In particular, the orderly cavities (Figure 25A, cavities numbered 1-4, toluene blue counterstaining, 10×) and the deposition of BioMag particles (brown) lining the through-cavities were evident (Figure 25B, 40×), indicating that the microneedles penetrated human skin. Furthermore, analysis of sections from living human explants stained with DAPI to identify cell nuclei and anti-HLA-DR to identify MHC class II+ antigen-presenting cells revealed high-density fluorescent granules deposited in the superficial epidermis and dermis, including several particles co-localized with class II+ antigen-presenting cells (Figure 25C, DAPI (blue), HLA-DR+ (red), and fluorescent particles (green), 40×).

[0097] These results further demonstrate that the CMC microneedle array described herein can effectively penetrate human skin and deliver integrated cargo (bioactive components), including insoluble granular material. These are consistent with the current primary objective of rational vaccine design: effective delivery of particulate antigens to antigen-presenting cells in human skin.

[0098] To further address microneedle array delivery in vivo, in vivo cutaneous delivery of particulate antigens was modeled by similarly applying a fluorescent particle-containing array to the dorsal surface of the ear of anesthetized mice. After 5 minutes, the patch was removed, and the mice resumed their normal activity. At 3 hours or 3 days, the skin of the ear and afferent region lymph nodes were analyzed for the presence of fluorescent particles. Consistent with human skin findings, the particles were evident in skin excised from the array application site (data not shown). Furthermore, at 3 days, a considerable number of particles were evident within the afferent region lymph nodes. Figures 26A and 26B show a considerable number of particles evident within the afferent region lymph nodes (Figure 26A, 10×), including clusters of particles closely associated with class II+ cells (Figure 26B, 60×), suggesting the presence of lymph node resident antigen-presenting cells to which the particles had migrated internally (Figure 26A, 10×).

[0099] To quantitatively evaluate the effect of needle geometry on cargo delivery using microneedle arrays, 3 A CMC microneedle array labeled with H tracer was constructed. CMC hydrogel was prepared as a model active ingredient with a final dry weight content of 25 wt% using 5% wt ovalbumin (5 g / 95 g OVA / CMC), and 0.1 wt% trypan blue, 3 0.5 × 10⁻¹⁶ H-thymidine (ICN Inc., catalog number 2406005) 6 dpm / 1 mg dry weight 3 The samples were traced with H tracer. From a single batch of labeled CMC hydrogel preparations, four batches were obtained, each containing several individual patches with pyramidal and sharp columnar needle geometric shapes. 3 An H-CMC microneedle array was fabricated. The patch was applied to a human skin explant as described above, exposed for 30 minutes, and then removed. The patch treatment area was tape-stripted to remove surface debris, and the area was cut using a 10 mm biopsy punch. The cut human skin explant disc was 3 The H content was determined by scintillation counting. 3The specific activity of the H-CMC microneedle patch material was determined to be 72,372 cpm / 1 mg dry weight. This specific activity was used to indirectly determine the amount of ovalbumin that reached and was retained in the skin. The obtained data are summarized in Table 1 below.

[0100] The types of patches tested were consistent across microneedle arrays (mean standard deviation 24–35%) and batches (mean standard deviation 7–19%). Intra-batch variability in both needle geometries was below the in-batch value, suggesting that the insertion process and target characteristics likely play a major role in successful transdermal material delivery and retention. Patch material retention data clearly demonstrate that microneedle geometry is of primary importance in transdermal cargo delivery. Columnar needle geometries were 3.89 times larger overall than pyramidal needles. 3 This resulted in the deposition of H-labeled needle material. Based on the deposited radioactive material, it is estimated that the pyramidal needles were inserted to a depth of approximately 200 μm, while the columnar needles were inserted to a depth of approximately 400 μm or more.

[0101] Table 4.2.5. Injection of human skin explants by pyramidal and columnar needles 3 Transfer of H-labeled CMC microneedle material [Table 1]

[0102] Preferably, the microneedle arrays described herein can be used for cutaneous immunization. Developing strategies for the effective delivery of antigens and adjuvants is a primary objective of vaccine design, and immunization strategies targeting cutaneous dendritic cells offer various advantages over traditional vaccines.

[0103] The microneedle arrays described herein may also be effective for chemotherapy and immunochemotherapy applications. Effective and specific delivery of chemotherapeutic agents to tumors, including skin tumors, is a major objective of modern oncology. However, systemic delivery of chemotherapeutic agents is limited by several well-established toxicities. Local delivery may be effective in the case of skin tumors, including cutaneous tumors (basal cell, squamous cell, Merkel cell, and melanoma, etc.) as well as metastatic tumors to the skin (breast cancer, melanoma, etc.). Current methods of local delivery generally require repeated application of cream or local injection. The effectiveness of these methods is currently limited by the limited penetration of the active ingredient into the skin, nonspecificity, and undesirable side effects.

[0104] The microneedle arrays of this disclosure can be used as an alternative to or in addition to traditional localized chemotherapy techniques. The microneedle arrays of this disclosure can penetrate the outer layers of the skin and effectively deliver active biological agents to living cells in the dermis and epidermis. Upon delivery of the chemotherapeutic agent, apoptosis and death of skin cells are induced.

[0105] Furthermore, multiple bioactive agents can be delivered in a single microneedle array (patch). This enables immunochemotherapy approaches based on co-delivery of cytotoxic agents with immunostimulants (adjuvants). In the immunogenic environment created by the adjuvant, tumor antigen release from dying tumor cells is presented to the immune system, inducing local and systemic antitumor immune responses that can reject tumor cells at the treatment site and throughout the body.

[0106] In exemplary embodiments, the delivery of biologically active small molecules was tested. In particular, the activity of the chemotherapeutic agent Cytoxan® delivered to the skin using a CMC microneedle array was tested. Cytoxan® represents a class of active ingredients with potential clinical utility for the topical treatment of a range of skin malignancies, and allows for the direct measurement of its biological activity (Cytoxan®-induced apoptosis in the skin).

[0107] To directly evaluate the immunogenicity of antigens incorporated into CMC microneedle arrays, well-characterized model antigens of ovalbumin were used. Pyramid arrays were constructed incorporating soluble ovalbumin (sOVA), granular ovalbumin (pOVA), or arrays containing both CpG and pOVA. The adjuvant effect of CpG has been well-characterized in animal models, and these adjuvant activities in humans are currently being evaluated in clinical trials.

[0108] Immunization was performed, as described above, by applying antigen-containing CMC microneedle arrays to the ears of anesthetized mice using a spring-loaded applicator, and then removing the arrays 5 minutes after application. These pyramidal microneedle arrays contained approximately 5 wt% OVA and approximately 0.075 wt% (20 μM) CpG in the CMC. A gene gun-based gene immunization strategy using plasmid DNA encoding OVA was used as a positive control. Gene gun immunization is one of the most potent and reproducible methods for inducing a CTL-mediated immune response in mouse models, suggesting its use as a “gold standard” for comparison in these assays.

[0109] Mice were immunized, boosted after one week, and then assayed in vivo for OVA-specific CTL activity. In particular, immunization with arrays containing small amounts of OVA and CpG induced high levels of CTL activity, similar to that observed by gene gun immunization (Figure 27). Significant OVA-specific CTL activity was induced with both granular and soluble array-delivered OVA antigens, even in the absence of adjuvants. It is well established that a similar response requires substantially higher doses of antigen when delivered by conventional needle injection.

[0110] To evaluate the stability of the fabricated arrays, batches of arrays were fabricated, stored, and then used over extended periods. As shown in Figure 28, no significant degradation of immunogenicity was observed over storage periods of up to 80 days (the longest time period evaluated). Therefore, CMC microneedle arrays and this delivery technology can enable effective skin delivery of antigens and adjuvants to induce antigen-specific immunity.

[0111] To evaluate the delivery of biologically active small molecules, pyramidal CMC microneedle arrays containing the low molecular weight chemotherapeutic agent Cytoxan® (cyclophosphamide), or FluoresBrite green fluorescent particles as a control, were fabricated. Cytoxan® was integrated at a concentration of 5 mg / 1 g CMC, allowing for delivery of approximately 140 μg per array. This is a therapeutically reasonable concentration based on the area of ​​skin targeted, but well below levels associated with systemic toxicity. The cytotoxicity of Cytoxan® was evaluated using organ cultures of living human skin. Cytoxan® was delivered by applying the array to skin explants, as previously described by the inventors. After 5 minutes and 72 hours of exposure, the array and residual material were removed, and the cultured living skin explants were freeze-sectioned and fixed. Apoptosis was evaluated using the green fluorescence TUNEL assay (In Situ Cell Death Detection Kit, TMR Green, Roche, catalog number: 11-684-795-910). As shown in Figure 29A, fluorescence microscopy image analysis of human skin sections revealed widespread apoptosis of epidermal cells in skin treated with Cytoxan®. As shown in Figure 29B, no visible apoptosis was observed in skin treated with fluorescent particles, but these particles were clear and validated that the observed extent was precisely targeted by the microneedle array.

[0112] Directly fabricated microneedle array The micro-milling of the master mold described above enables the production of microneedle arrays having various geometric shapes. In another embodiment, a system and method are provided for producing microneedle arrays by directly micro-milling various materials such as dried CMC sheets. The same general molding tools described above for micro-milling of master molds can be used to directly micro-mill microneedle arrays.

[0113] Direct micromilling of microneedle arrays eliminates the need for molding steps, enabling a simplified, scalable, and highly repeatable production strategy suitable for large-scale clinical applications. Furthermore, direct fabrication of microneedle arrays through micromilling allows for greater control over microneedle geometry. For example, micromilling makes it possible to include microneedles that retain features such as undercuts and / or bevels, which cannot be achieved using molding processes.

[0114] The reproducibility of direct milling of microneedle arrays is particularly beneficial. That is, in direct micromilling, all microneedles are identical as a result of the milling process. In molding operations, it is not uncommon for some needles to be missing or broken from a given patch as a result of the process of physically separating them from the mold. For use in certain medical applications, the reproducibility of the amount of bioactive ingredient in the array is crucial to providing an appropriate level of "quality control" across the process, because needle variability between patches would likely result in variability in the dose of drug / vaccine delivered. Of course, reproducibility is also a significant advantage for any application requiring FDA approval. Spin-cast / molded patches would require a special process to ensure acceptable uniformity for consistent drug delivery. This quality control would also likely result in a certain percentage of patches "failing" this release test, introducing waste into the production process. Direct micromilling eliminates, or at least significantly reduces, these potential problems.

[0115] The molding process also has inherent limitations due to the need to fill wells or concaves and to be able to remove the cured molded part from those wells or concaves. That is, due to the geometry of the mold, undercuts must generally be avoided when molding a part, otherwise the part will not be removable from the mold. In other words, a geometric limitation of the molded part being molded, such as a microneedle array, is that any feature located closer to a vertex must be narrower than any feature located toward the base.

[0116] Therefore, taking these limitations into consideration, Figure 20 shows a schematic diagram of a microneedle shape and structure generally suitable for fabrication by molding. That is, the shape and structure shown in Figure 20 does not contain any undercuts that would prevent the part (i.e., the microneedle) from being removed from the manufacturing mold. In contrast, Figure 30 shows a microneedle shape with oblique undercuts that cannot be molded in the manner described herein.

[0117] This geometry can only be fabricated by direct production using the proposed micromilling technique. The negative (bevel) angle promotes better retention of the microneedle within the tissue. Furthermore, since the microneedle in Figure 30 has a wider middle section (with a larger cross-sectional dimension) above the lower section (with a smaller cross-sectional dimension), a larger amount of bioactive material can be delivered by configuring the microneedle to hold or store the bioactive material in the wider section configured to be retained within the skin. Thus, the larger cross-sectional dimension of the middle section can "carry" the bulk of the bioactive component. As the lower section tapers towards a narrower cross-sectional dimension, the wider middle section will have good penetration for delivering the bioactive component into the skin layer. The upper portion of the middle section should preferably taper towards the tip to facilitate the penetration of the microneedle into the skin layer.

[0118] Another limitation of molded parts is that it can be difficult to accurately fill very small sections of the mold. Since manufacturing molds for microneedle arrays contain numerous very small sections, it can be difficult to accurately fill each well. This can be particularly problematic when the mold must be filled with different materials, such as materials containing bioactive components and materials that do not. Therefore, when the manufacturing mold is filled in layers, it can be difficult to accurately fill the very small wells associated with each microneedle. Such reproducibility is particularly important because microneedles are intended to deliver one or more bioactive components. Therefore, even slight variations in the amount of bioactive component used to fill the manufacturing mold can be highly undesirable.

[0119] Furthermore, by using a layered structure to form a sheet or block that can be micromilled, various active ingredients can be integrated into a single microneedle by vertical layering. For example, in an exemplary embodiment, CMC hydrogel and CMC-sOVA hydrogel (80% CMC / 20 wt% OVA) were layered to form a sheet or block. This composite sheet can be micro-machined using the direct micromilling technique described herein.

[0120] Figure 31 shows a stereomicroscopic image analysis of the entire microneedle array. The microneedle array consists of 10 × 10 microneedles. Figure 32 is a magnified segment of the microneedle array from Figure 31. The stratification of two components is shown in Figure 32, which indicates a darker region of the microneedles at the tip and a brighter region of the microneedles at the base. The darker layer at the tip represents the layer containing the bioactive component, in this case, soluble ovalbumin contained in the CMC layer.

[0121] The formation of a layer containing an active material (e.g., an antigen), and subsequent micromilling of this layer (and any other adjacent layers), may require the use of a relatively large amount of active material, but the material can be removed (e.g., in the form of a tip), recovered, and reused. Direct mechanical cutting techniques are not limited by the geometric constraints arising from molding / demolding methods, and therefore can create more innovative needle designs (e.g., Figure 30), which can significantly improve the volume of the needle retained in the skin and the needle retention time.

[0122] By producing sheets or blocks by forming multiple layers, it is possible to obtain solid materials that can be micro-machined and may contain one or more layers with bioactive components. For example, soluble solid carboxymethylcellulose polymer-based blocks or sheets with well-defined, controlled dimensions can be fabricated by a lamination process. The resulting sheets or blocks are fully machineable, similar to the machining of plastic or metal sheets or blocks. As described herein, this fabrication process may be suitable for incorporating bioactive components into the matrix without significantly reducing their activity levels.

[0123] As described below, fabricated sheets of material (such as CMC-based material) can be directly micro-machine-cut / micro-milled to produce one or more microneedle arrays suitable for delivering active components through the skin. This soluble biocompatible CMC block material can be used to deliver soluble or insoluble and particulate active substances in a sustained-release manner for application to the body surface.

[0124] Biocompatible materials may be suitable for implants in deeper soft or hard tissues, where dissolution of scaffolding materials is required and where useful.

[0125] The following method can be used to prepare a 12.5% ​​carboxymethylcellulose (CMC) polymer low viscosity hydrogel. A 12.5% ​​carboxymethylcellulose (CMC) low viscosity hydrogel can be prepared in water or (but not limited to) other biocompatible buffers such as PBS or HBS. During the preparation of the polymer solution, soluble active agents (such as nucleic acids, peptides, proteins, lipids, or other organic and inorganic biologically active components), as well as granular materials (e.g., ovalbumin, soluble active agent), can be added. Ferrous granular materials supporting the active components at 20 w / w% of the CMC can be used.

[0126] The preparation of 1000g of sterile 12.5% ​​CMC hydrogel completely free of active ingredients can be achieved as follows: 1) Measure 125g of CMC and add 875g of water or another water-based solvent. 2) Mix in an overhead mixer until homogenized. 3) Sterilize the homogenate by autoclaving at 121 degrees Celsius for 1 hour (autoclaving can reduce viscosity and improve stratification). 4) Cool to 22 degrees Celsius. 5) The obtained material is vacuum-treated at 10 Torr and 22°C for 1 hour to remove trapped microbubbles. 6) Centrifuge the product at 25,000 g for 1 hour in a centrifuge with a vacuum chamber (to float and further remove any remaining microbubbles). 7) Store the CMC hydrogel product at 4 degrees Celsius.

[0127] The preparation of 1000 g of sterilized 20 / 80% ovalbumin / CMC hydrogel with a dry content of 12.5 w / w% can be achieved as follows: 1) Measure 100g of CMC and add 650g of water or other water-based solvent. 2) Mix in an overhead mixer until homogenized. 3) Sterilize the homogenate by autoclaving at 121 degrees Celsius for 1 hour (this autoclaving step can reduce viscosity and improve stratification). 4) Cool to 22 degrees Celsius. 5a) Dissolve 25g of ovalbumin in 225g of water. 5b) Sterile filter the ovalbumin solution using a filter with a pore size of 0.22 μm. 6) Mix 750g of CMC hydrogel with 250g of sterile ovalbumin solution under sterile conditions until homogeneous. 7) The obtained material is vacuum-treated at 10 Torr and 22°C for 1 hour to remove trapped microbubbles. 8) Centrifuge the product at 25,000 g for 1 hour in a centrifuge with a vacuum chamber (to float and further remove any remaining microbubbles). 9) Store the CMC hydrogel product at 4°C.

[0128] The preparation of 100 g of sterile granular ovalbumin / CMC hydrogel with a dry content of 12.5 w / w% (20 / 80%) can be achieved as follows: 1) Measure 10 g of CMC and add 87.5 g of water or other water-based solvent. 2) Mix in an overhead mixer until homogenized. 3) Sterilize the homogenate by autoclaving at 121 degrees Celsius for 1 hour (this autoclaving step can reduce viscosity and improve stratification). 4) Cool to 22 degrees Celsius. 5) Disperse 2.5 g of granular ovalbumin in 97.5 g of CMC hydrogel at 22 degrees Celsius and mix under sterile conditions until homogeneous. 6) The obtained material is vacuum-treated at 10 Torr and 22°C for 2 hours to remove trapped microbubbles. 7) Centrifuge the product at 3,000g for 1 hour in a centrifuge with a vacuum chamber (to float and further remove any remaining microbubbles). 8) Store the CMC hydrogel product at 4°C.

[0129] In this example, granular ovalbumin is prepared by the reaction of activated iron beads with ovalbumin. However, it should be noted that the above description is merely an illustrative embodiment, and other compounds and active components may also be used.

[0130] Solid blocks / sheets of carboxymethylcellulose (CMC) can be manufactured using the low-viscosity CMC hydrogel described above in the following manner.

[0131] This fabrication process may include layering and diffusing a polymer to a specified thickness, and then drying the layered polymer to a moisture content of less than approximately 5% using a sterile, dry airflow on the surface of the polymer layer. These two actions can be repeated until the desired block thickness is achieved.

[0132] A method for achieving layered CMC hydrogel layering of a specified thickness in a mold assembly will be described with reference to Figure 33. Figure 33 shows a cross-sectional view of a mold assembly including (a) a casting bed; (b) an adjustable casting bed wall; (c) a casting bed depth adjustment assembly; and (d) an acrylic sprayer. It should be noted that Figure 33 is not drawn to a constant scale, nor is it shown with elements in appropriate proportions in any other way.

[0133] The mold assembly can be constructed from acrylic (plexiglass) and may include a casting bed base unit, a vertically adjustable hydrophobic casting bed wall, and a casting bed adjustment mechanism. The casting bed base unit (a1) may include a removable / replaceable casting bed top plate (a2) to which a cellulose layer (a3) ​​is attached. The cellulose layer may be approximately 0.5 mm thick. The vertically adjustable hydrophobic casting bed wall (b) can be adjusted using a casting bed depth adjustment mechanism which may consist of a lead screw (c1) and a level adjustment knob (c2). In the embodiment shown, turning this knob a quarter turn raises the bed wall by 0.5 mm.

[0134] First, the adjustable casting bed wall can be set to a height where the distance between the acrylic sprayer and the cellulose layer of the bed is approximately 1 mm when the sprayer is in the correct position. (For example, approximately 0.1 ml / cm³) 2 ) 12.5% ​​CMC hydrogel can be added and layered. The layer can be leveled or flattened by sliding an acrylic sprayer (d) on the upper surface of an adjustable casting wall to obtain a leveled layer of CMC hydrogel of approximately 1 mm. The layered CMC hydrogel can be dried to a solid phase in a drying apparatus shown in Figure 34 and described in more detail below.

[0135] The layering and drying steps can be repeated until the desired layered structure (sheet) is achieved. The casting bed wall can be raised by an appropriate amount between each layer addition. For example, after each layer is added, the bed wall can be raised or lifted by approximately 0.5 mm. Thus, the cycle described above can deposit a solid CMC layer of approximately 0.5 mm. This process (e.g., layering of material, raising of the bed wall, etc.) can be repeated until the desired block thickness is achieved.

[0136] Layered CMC hydrogel polymers can be dried in various ways. For example, Figure 34 shows a drying apparatus that can be used to dry various deposited layers of sheet material. It should be noted that Figure 34 is not drawn to a constant scale or otherwise shown with elements in appropriate proportions. A fan can bring a continuous gas flow (e.g., air or other inert gas such as nitrogen) onto the layered CMC hydrogel within the mold assembly. The gas flow gently dehydrates the CMC hydrogel layer. The drying rate can be adjusted to prevent or reduce gas inclusion (e.g., bubbles) in the solid CMC product. High-humidity air on the layer can be dried with a desiccant (e.g., an air dryer or dehumidifier), the temperature adjusted, and returned to the hydrogel by a speed-controlled fan. A hygrometer can be placed in position on the high-humidity side of the chamber to indicate the state of the drying process. When indicated by the hygrometer, the drying process can be terminated after a predetermined level of drying has been achieved.

[0137] The airflow can be adjusted to affect the drying rate. In an exemplary embodiment, the airflow is controlled between approximately 0.1 and 2.0 m / s, and the temperature is between the ambient temperature and approximately 50 degrees Celsius. Using these configurations, the drying time for a single-layer CMC hydrogel can be approximately 0.5 to 4 hours, depending on the airflow and set temperature.

[0138] Pure CMC-based products can be clear, pale off-white, or amber in color. Their specific gravity can be approximately 1.55–1.58 g / ml. The product should ideally be free of microbubbles and otherwise suitable for fabricating micron-scale objects. The physical characteristics of the final block / sheet product (hardness, tensile strength, etc.) can vary, but generally, it should be able to withstand the physical stresses associated with micromilling.

[0139] As described above, the microneedle arrays disclosed herein can provide a reliable and precise method for the delivery of various bioactive components. The characteristic structural, manufacturing, and distribution advantages of the microneedle arrays described above may be particularly applicable to use in vaccine delivery. The advantages of these microneedle arrays include (1) safety due to the elimination of the need for needles or live vectors for vaccine delivery, (2) economics due to low production costs, product stability, and ease of distribution, and (3) versatility through a delivery platform compatible with a variety of antigens and adjuvant formulations.

[0140] Furthermore, immunization of the skin with microneedle arrays offers significant advantages in terms of immunogenicity. Skin is rich in readily accessible dendritic cells (DCs) and has long been considered a highly immunogenic target for vaccine delivery. These dendritic cell populations constitute the most potent antigen-presenting cells (APCs) identified to date. For example, genetic immunization of the skin transfects and activates mouse and human skin dendritic cells. These transfected dendritic cells synthesize transgenic antigens, migrate to lymph nodes in the skin inflow area, and efficiently present these antigens via MHC class I-restricted pathways to stimulate CD8+ T cells. Immune responses induced by skin-derived DCs are remarkably potent and persistent compared to those induced by other immunization methods. Recent clinical trials have demonstrated that even conventional vaccines are significantly more potent when delivered intradermally rather than by standard intramuscular needle injection. Therefore, microneedle arrays can efficiently and simultaneously deliver both antigens and adjuvants, enabling both DC targeting and adjuvant engineering of the immune response using the same delivery platform.

[0141] High-frequency electromagnetic vibration applicator Microneedle array devices can be applied to human skin by various methods, including self-application or assisted application using human pressure (e.g., pressing with a finger or thumb) or a spring-loaded device. To facilitate the ease and reproducibility of delivery of microneedle array devices, including tip-loaded microneedle arrays, an applicator device is described herein. The applicator device is configured to convert high-frequency electromagnetic vibrations into unidirectional mechanical resonances of an active head. This, in turn, enables multiple reproducible low-amplitude and high-frequency pressure strokes, which facilitate the insertion of microneedles of the microneedle array into tissue, including human skin.

[0142] As shown in Figure 38, the applicator may comprise an applicator head, an oscillator-energy converter, an electromagnetic oscillator, and a power supply. If necessary, one or all of these four elements may be separable from the applicator device.

[0143] Applicator heads can be made interchangeable to adapt to and act on tissue surface areas of different sizes and shapes. As shown in Figure 39, various applicator head geometries can be used in combination with the applicators described herein. Applicator heads are interchangeably fabricated from stainless steel or other chemically and physically resistant materials. If necessary, applicator heads can be autoclaved and / or sterilized with alcohol or other chemicals for sterilization. Gas sterilization (ethylene oxide) is possible as an alternative or additional method.

[0144] Application-specific geometries can be quickly designed and manufactured. For example, the area of ​​a single application in this embodiment is 5 mm depending on the geometry of the active head. 2 ~250mm 2 This range can be achieved through simple structural variations in the geometric shape of the head.

[0145] The oscillator energy converter unit can be configured to convert electromagnetic vibrations into mechanical motion of the applicator head. The amplitude of the applicator head in direction Z can be controlled between 0.5 and 2.5 mm (Figure 40; A). In some embodiments, the head motion in directions X and Y can be configured to be negligible, i.e., less than 0.2 mm (Figure 40; B). The frequency of the mechanical motion resulting from the energy conversion in direction Z can be controlled between 500 and 25,000 rpm. If necessary, the oscillator energy converter unit may be separable and can be disposed of or sterilized as required.

[0146] An electromagnetic (EM) oscillator may consist of three subunits. These subunits may include (1) a control unit and a regulating power supply that generates voltage and power for the high-frequency EM oscillator, (2) a control unit-regulator that generates a high-frequency signal and required current for the EM oscillator, and (3) the EM oscillator itself. The output frequency can be controlled by the user (e.g., within a range of 100-500 Hz). In some embodiments, the EM oscillator may be completely sealed and can be sterilized with an alcohol solution or other chemical agent.

[0147] The power supply unit also has different compatible power supplies, for example: a. Regular disposable alkaline or any other type of battery, b. Rechargeable NiCad or Li-oxide battery with a built-in induction charger, c. 100~240V electronic power adapter It may be possible to separate it to adapt to various situations.

[0148] Applicators can offer several advantages when used with microneedle arrays. For example, they can minimize the mechanical force required to insert the microneedle array into the tissue. Applicators can also reduce pain compared to existing spring-loaded applicators. Furthermore, applicators can be portable, and their components can be separable and interchangeable. Finally, applicators can be configured to be sterilizable for sterile use.

[0149] Given the many possible embodiments to which the principles of the disclosed embodiments may be applied, it should be recognized that the embodiments shown are merely preferred examples and should not be interpreted as limiting the scope of protection. Rather, the scope of protection is defined by the following claims. Accordingly, the inventors claim all that falls within these claims and their intent.

Claims

1. A microneedle array for treating skin cancer in a target skin region, It's cargo, A soluble biocompatible material containing a dehydrated carboxymethylcellulose hydrogel layer, and One or more bioactive components in a therapeutically effective amount incorporated into the dehydrated carboxymethylcellulose hydrogel layer The cargo includes, and the one or more biologically active components include doxorubicin in an amount of 100 μg per microneedle array and at least one viral vector, Base, and Multiple microneedles extending from the base Includes, The plurality of microneedles include an upper half, a lower half, and an intermediate portion located between the upper half and the lower half. The aforementioned plurality of microneedles taper from the middle portion to a certain point in the upper half, The aforementioned point has a vertex angle greater than 30 degrees, The cargo is positioned within the upper half of the plurality of microneedles, and the base does not contain any bioactive components. The microneedle array is configured to be inserted into the target skin region of the subject, and the microneedle array is configured to penetrate the stratum corneum and deliver one or more of the bioactive components to the epidermis and / or dermis by dissolving the microneedles, thereby providing the subject with a therapeutically effective amount of one or more of the bioactive components through local skin delivery without systemic exposure. Microneedle array.

2. The microneedle array according to claim 1, wherein the one or more biologically active components of the microneedle array include at least two different chemotherapeutic agents.

3. The microneedle array according to claim 2, wherein the at least two chemotherapeutic agents include a cytotoxic agent and an immunostimulant.

4. The microneedle array according to claim 3, wherein the immunostimulator comprises at least one adjuvant.

5. The microneedle array according to claim 1, wherein the one or more biologically active components are selected from the group consisting of chemotherapeutic agents, adjuvants, and chemoattractants for cancer chemoimmunotherapy.

6. The microneedle array according to claim 1, wherein the at least one viral vector includes an adenovector.

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