RU2816629C2 - Microneedle matrices with undercut elements for dermal and non-dermal drug delivery - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to medical equipment, namely to methods of forming a microneedle matrix from a production injection mould, which contains a flexible material for moulds. Production injection mould includes multiple cavities, which are shaped to define multiple corresponding microneedles, each of which has core, microneedle point, rounded base and at least one undercut element. Flexible material has sufficient elasticity to allow extraction of the moulded microneedle matrix from the production mould in one drawing without damaging the integrity of the microneedle mould defined by the mould, as well as with preservation of mould integrity during multiple applications. Microneedles can have many different geometrical designs and can include different soluble and insoluble layers, as well as different types of substrates.

EFFECT: disclosed method can be used for reproducible production of soluble microneedle matrices with a coating and with undercuts and rounded bases, as well as insoluble microneedle matrices with undercuts to be used as adhesive applicators on tissue.

45 cl, 11 dwg

Description

CROSS REFERENCE TO RELATED APPLICATION

[001] This application claims priority to U.S. Provisional Application No. 62/848,939, filed May 16, 2019, which is incorporated herein by reference in its entirety.

FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

[002] The present invention relates to microneedle arrays and, in particular, to microneedle arrays formed with undercut features for so-called dermal and non-dermal drug delivery, as well as for adhesive applicators on body tissue.

NOTICE OF GOVERNMENT SUPPORT

[003] The present invention was made with government support under Grant Nos. AR071277 and AR074285 from the National Institutes of Health. The government has certain rights to an invention.

BACKGROUND OF THE INVENTION

[004] Leather is a readily available tissue with many different functions, such as a protective barrier and temperature regulator. It is worth noting that the skin acts as an active organ of the immune system. For some purposes, such as immunization, cancer immunotherapy, and allergy vaccination, the skin may be a preferred target anatomical site because it contains large populations of professional antigen-presenting cells and immune system A cells. Most vaccines, immunomodifiers, and anticancer drugs are administered using injections with hypodermic needles. However, drug delivery using traditional syringes has several disadvantages. These disadvantages include the need for trained health care personnel to administer, trypanophobia (i.e., fear of needle sticks), poor patient compliance with instructions for taking the drug, the risk of disease transmission and injury to health care workers from the needle, and the costs of storage and transportation in the cold chain system. .

[005] In addition, parenteral injections are unable to reproducibly and accurately deliver bioburdens to target skin microenvironments. Therefore, vaccines and drugs administered through conventional injections may result in insufficient effectiveness. Collectively, these factors impede the effective use of vaccines, antineoplastic drugs, or immunomodulators and result in ineffective cutaneous immunization and treatment strategies.

SUMMARY OF THE INVENTION

[006] This application discloses various methods and systems related to the manufacture of microneedle arrays and, in particular, microneedle arrays formed with undercut features for dermal and non-dermal drug delivery, as well as for adhesive applicators on body tissue.

[007] In some of the embodiments described below, a method for forming a microneedle array comprises the step of forming a production injection mold of a flexible material containing a plurality of cavities that are shaped to define a plurality of corresponding microneedles, each of which has a shaft, a microneedle tip, a rounded base, and at least one undercut feature, the step of incorporating at least one bioactive material within the first soluble material to provide a biodegradable matrix, the step of delivering the biodegradable matrix to at least a microneedle tip region defined by respective cavities of the production injection mold, the step of forming a plurality of microneedles in the production injection mold form, which include a biodegradable matrix, and the step of removing the microneedles from the production injection mold by drawing the microneedles from the injection mold. The flexible material may have sufficient elasticity to allow the molded microneedle array to be removed from the production injection mold in a single pull without damaging the integrity of the microneedle shape defined by the injection mold.

[008] In other embodiments, a microneedle array is formed using a pull-release injection mold containing a substrate, a plurality of microneedles, and at least one bioactive material combined with the first soluble material to form a biodegradable matrix. The plurality of microneedles contains, in each microneedle, a shaft, a microneedle tip, a rounded base and at least one undercut element.

[009] In yet other embodiments, a method of forming an injection molding matrix comprises the step of creating a 3-dimensional computer-aided design drawing (3D CAD drawing) of a microneedle array that includes a plurality of microneedles with at least one undercut feature, the step of forming a basic microneedle array using a 3D CAD drawing, the step of forming at least one replica of the base microneedle array, and the step of forming a production injection mold of the microneedle array using at least one replica. A production injection mold is formed from a flexible material.

[010] The above and other objects, features and advantages of the invention will become apparent from the following detailed description taken by reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[011] FIG. 1 is a depiction of exemplary microneedle designs that have, in the examples provided, pointed conical tips, round undercut shafts, and rounded bases.

[011] Figures 2A-C are images of a microneedle array that is formed with a compliant substrate.

[013] FIG. 3A is a computer-aided design (CAD) drawing showing exemplary MNAs that include sharp needles with undercut features and rounded bases.

[014] FIG. 3B is a 3D CAD drawing showing an exemplary MNA with a 5x5 needle pattern.

[015] FIG. 4A is a flow chart of an exemplary MNA manufacturing process.

[016] FIG. 4B illustrates various steps of the exemplary MNA fabrication process shown in FIG. 4A.

[017] Figure 5A is an optical microscope image of a basic MNA created using direct 3D laser writing.

[018] Figure 5B is an optical microscope image of a base MNA replica created using a two-step micromolding strategy.

[019] FIG. 5C is an optical microscope image of microneedle-shaped loading cavities in production MNA injection molds.

[020] Figure 5D is an optical microscope image of a single undercut needle on a 3D printed base MNA.

[021] Figure 5E is an optical microscope image of a single undercut needle on a replica of a base MNA.

[022] Figure 6A is an optical microscope image of the final dissolving MNAs from (CMC (carboxymethylcellulose)/Treh (trehalose)) (CMC/Treh-MNA), including bioburdens (for example, OVA (ovalbumin) + Poly(I :C) (copolymer of polyinosinic and polycytidylic acids)).

[023] Figure 6B is an optical microscope image of dissolving PVP (polyvinyl pyrrolidone)/PVA (polyvinyl alcohol) MNAs (PVP/PVA-MNA) with drug-containing tips and undercut microneedles incorporating OVA.

[024] FIG. 6C is an optical microscope image of a single CMC/Treh microneedle with a drug-containing tip and undercut, incorporating doxorubicin as a colored drug to facilitate visualization.

[025] Figure 7A is an image of PVP/PVA matrices incorporating dextran (about 40 kDa molecular weight) stained with Texas red on microneedle tips.

[026] Figure 7B is an image of CMC/Treh matrices with drug tips incorporating Allura Red R40 dye (with a molecular weight of about 500 kDa) in the pyramidal region of the microneedles.

[027] FIG. 7C is an image of PVP/PVA matrices with drug-containing tips including multiple loads such as Texas Red-stained dextran and Allura Red R40.

[028] FIG. 7D is an image of PVP/PVA matrices with drug-loaded tips including multiple loads such as Texas Red-stained dextran and PLGA (polylactide-co-glycolide) microparticles (average diameter 10 μm), tagged with Alexa488.

[029] FIG. 8A is an illustration of different microneedle designs fabricated from IP-S photoresist using direct 3D laser writing.

[030] Figure 8B is an image of printed microneedles with rounded bases.

[031] FIG. 9A is an optical microscope image of PVP/PVA matrices incorporating Allura Red before application to human skin.

[032] Figure 9B is a bright field microscope image of Allura Red R40 microneedle traces on living human skin samples.

[033] FIG. 9C is an optical microscope image of PVP/PVA-MNA incorporating Allura Red dye after application to human skin.

[034] Figure 9D-I is an image of intradermal co-delivery of Alexa488-labeled Poly(I:C) and Alexa555-labeled OVA from CMC/Treh-MNA with drug-loaded tips. Optical magnification 20x. Composite fluorescence microscope images demonstrate delivery cavities penetrating the epidermis and upper dermis and delivery of both antigen and adjuvant to targeted skin microenvironments.

[035] FIG. 10A is an optical microscope image of MNAs comprising both OVA with Alexa555 and Poly(I:C) with Alexa488, with a fluorescence image representative of a pyramidal tip containing both loads.

[036] Figure 10B is representative optical microscope images of MNAs containing OVA with Alexa555 and Poly(I:C) with Alexa488 after in vivo application to the depicted mouse.

[037] FIGS. 10C and 10D depict efficient co-delivery of Poly(I:C) with Alexa488 and OVA with Alexa555 into skin microenvironments using novel MNAs.

[038] FIG. 11A is representative histograms obtained from flow cytometry analysis showing residual CFSE (carboxyfluorescein succinimidyl ester)-labeled cells in the spleens of immunized and non-immunized mice.

[039] FIG. 11B is a quantitative representation of specific cell lysis, with 100% lysis corresponding to complete elimination of target cells (mean ± standard deviation, 3 mice per group).

[040] Figure 11C - Serum concentrations of specific antibodies IgG1 and IgG2c to OVA (bars represent average values, 3 mice per group).

DETAILED DESCRIPTION OF THE INVENTION

[041] The detailed descriptions herein describe certain exemplary embodiments relating to the manufacture and use of microneedle arrays (MNAs). Although exemplary embodiments may disclose specific types of MNAs, it should be understood that other types of MNAs may benefit from the disclosed systems and methods.

[042] As used herein, the term “biologic,” “active ingredient,” “bioburden,” or “bioactive material” refers to pharmaceutically active substances such as analgesics, anesthetics, antiasthmatics, antibiotics, antidepressants, antidiabetics, antifungal agents, antihypertensive agents, anti-inflammatory agents, antineoplastic agents, anxiolytic agents, enzyme-active agents, nucleic acid constructs, immunostimulants, immunosuppressive agents, vaccines and the like. The bioactive material may contain soluble materials, insoluble but dispersible materials, natural or formulated macro-, micro- and nanoparticles and/or mixtures of two or more soluble, dispersible insoluble materials and natural or formulated macro-, micro- and nanoparticles. nanoparticles In this regard, although a number of examples of MNAs disclosed herein relate to vaccines and immunizations, any other suitable bioactive material, such as the materials described above, can be used in the designs of novel MNAs.

[043] As used herein, the expression "preformed" means that the structure or element is manufactured, created and/or formed into a specific shape or configuration prior to use. Accordingly, the shape or configuration of the preformed microneedle array is the shape or configuration of the microneedle array prior to insertion of one or more microneedles of the microneedle array into a patient.

[044] As used herein, the term "undercut" or "undercut feature" refers to a recessed surface that is considered inaccessible using standard forming techniques and, in particular, to a structural feature (e.g., a recess, protrusion, or other geometric shape) which limits or prevents the molded part containing the element from being removed from a conventional one-piece mold.

[045] The systems and methods described herein and their individual components should not be construed as being limited in any way to the specific applications or systems described herein. Rather, the present disclosure relates to all new and non-obvious features and aspects of the various disclosed embodiments, individually and in various combinations and subcombinations with each other. For example, any features or aspects of the disclosed embodiments may be used in various combinations and subcombinations with each other, as would be recognized by one of ordinary skill in the relevant art(s), taking into account the information disclosed herein. Moreover, the disclosed systems, methods, and components thereof are not limited to any particular aspect or feature or combinations thereof, nor do the disclosed features and methods require that at least one of the particular advantages be presented or that one of the particular problems be presented solved. Headings are provided for readability purposes only, and it should be understood that elements and/or steps from one section may be combined with elements and/or steps described under other headings in this disclosure.

[046] As used herein, singular forms (expressed by articles) include plural forms unless the context otherwise clearly indicates. The expression "includes" means "contains". Further, as used herein, the expression “and/or” means any single element or combination of elements of a phrase. In addition, the definition of “exemplary” means a non-limiting example, particular case, or illustration. As used herein, the expressions “for example” and “for example” include a list of one or more non-limiting embodiments, examples, specific cases and/or illustrations.

[047] Although the operations of some of the disclosed methods are described in a particular sequential order for convenient presentation, it should be understood that this manner of description involves permutations unless a particular order is provided by the specific language below. For example, operations described in sequential order may in some cases be rearranged or performed simultaneously. Additionally, for simplicity, the accompanying figures may not show how the disclosed features and methods may be used in conjunction with other features and methods. Additionally, the specification sometimes uses expressions such as “provide,” “produce,” “specify,” and “select” to describe the method disclosed. These expressions are high-level descriptions of the actual operations that are performed. The actual operations that correspond to these expressions will vary depending on the particular implementation and will be readily apparent to one of ordinary skill in the art taking advantage of the present invention.

[048] Exemplary Microneedle Arrays

[049] The use of microneedle arrays offers numerous advantages over conventional needle injection methods. For this reason, cutaneous vaccination or drug delivery using a microneedle array offers a reliable and feasible approach for effective immunization or immunotherapy of cancer due to the above-mentioned theoretical beneficial properties of skin.

[050] In contrast to topical delivery methods, MNAs physically penetrate the stratum corneum of the epidermis, which eliminates the complexities of drug formulation and leads to local accumulation of vaccines or drugs in the skin microenvironment. In contrast to injections with traditional needles, microneedles are gentle on pain receptors, allowing for minimally invasive, painless immunization.

[051] Among other advantages, dissolving MNAs can provide highly effective vaccination due to their higher antigen capacity, controlled release kinetics, ease of manufacture and long-term stability. Such MNAs can be created from water-soluble polymers that dissolve when injected into the skin. The microneedles in the MNA are preferably strong enough in their dry state to be embedded in the stratum corneum of the epidermis and then quickly dissolve in the fluid environment of the skin, thereby releasing the vaccines. Precise delivery of bioactive materials, such as vaccines, into the skin microenvironment may result in increased efficacies, resulting in relatively lower doses required compared to traditional needle injections.

[052] As described in more detail below, microneedles with undercut geometries that include soluble and/or insoluble materials can be formed using the new methods and systems described herein.

[053] In some embodiments, new MNAs can be formed through a one-step micromolding process that uses flexible materials for a production injection mold. The use of such flexible materials in production injection molds enables the production of a variety of geometric designs that would not otherwise be possible. The flexible material for the production injection mold may comprise, for example, any elastomer or other flexible material that provides removable microneedles of the desired design (eg, with the desired amount of undercut and/or other geometric shape). The ability to mold a variety of geometric shapes that are otherwise difficult to produce by molding makes possible new and advanced functionally graded MNAs, such as those shown in Fig. 1, for targeted delivery of many different bioactive materials to the skin and other tissues (eg, cardiac and ocular tissue).

[054] Figure 1 depicts examples of various microneedle designs that can be formed using the methods and systems described herein. 1 shows four different microneedle designs 10, 12, 14, 16 in a production injection mold 18 that is formed from a flexible material, such as a flexible elastomer.

[055] The microneedle 10 is formed with a soluble substrate 20, a soluble rod 22, and a microneedle tip 24, which is filled with a bioactive material. Thus, the microneedle 10 is made entirely of dissolving (or biodegradable) material. The bioactive material can be mixed into the dissolving material, but is preferably located on the needle point, as shown in Fig. 1, to improve delivery efficiency.

[056] As shown in FIG. 1, the microneedles 10, 12, 14, 16 may have a pyramid-shaped tip with a pointed tip and an undercut shaft that connects to the substrate through a rounded base. The fillets 32 may provide improved mechanical properties during insertion into tissue.

[057] The MNAs described herein can be made from any moldable dissolving, biodegradable, and/or biocompatible insoluble materials, including carboxymethylcellulose, trehalose, polyvinylpyrrolidone, polyvinyl alcohol, maltodextrin, silk, glucose, hyaluronic acid, polymethyl methacrylate, polycarbonate, copolymer of lactic acid and glycolic acid, polylactic acid, fast-curing resins and combinations thereof to include any bioactive materials, including cosmetics, dermal fillers, statins, growth factors, analgesics, antihistamines, vitamins, anesthetics, anti-aging agents, small molecule drugs, haptens, allergens, anti-inflammatories, proteins, peptides, microbubbles, exosomes, polyplexes (small interfering RNA, short hairpin RNA, DNA vector complexes), recombinant viral vectors (i.e. adenovirus, lentivirus, vaccinia virus, adeno-associated virus and their various serotypes), monoclonal and polyclonal antibodies and live or lysed cells.

[058] The microneedle 12 is formed with an insoluble support 26, an insoluble rod 28, and a microneedle tip 24, which is filled with a bioactive material. These functionally graded undercut MNA designs can be made from at least two materials. For example, the pyramidal portion can be created using a dissolving or biodegradable material, and the core portion and backing are made from an insoluble material, such as an insoluble biocompatible rigid polymer (eg, polymethyl methacrylate, polycarbonate, VeroWhite polymer, and other ultraviolet-curable (UV-curable and thermosetting resins) ).

[059] Therefore, the microneedle 12 can provide a sharp needle point, together with enhanced mechanical properties due to the rounded base, the ability to effectively penetrate tissue, and a pyramidal tip that serves as a dosage form of the bioactive material in which the bioactive material(s) are included into a matrix of dissolving or decomposing biomaterial. The undercut core portion enhances mechanical performance during embedding while providing tissue retention during implantation, and the non-dissolving undercut core portion prevents back diffusion of the embedded bioactive material(s) during both manufacturing and manufacturing processes. implantation The insoluble support may also help prevent moisture absorption during storage, which can cause excessive curvature of the supports and reduce the effectiveness of MNA applications.

[060] The microneedle 14 is similar to the microneedles 12, 14 in shape, but additionally includes another dissolving layer 30. For example, a pyramidal portion is created using a dissolving or biodegradable material, and a faster dissolving layer is provided adjacent the connection of the shaft to the needle tip. The dissolution layer may be formed, for example, from a rapidly dissolving low molecular weight polymer such as glucose, sucrose, trehalose, maltodextrin, or polyvinylpyrrolidone. The remainder of the core portion and the support may be formed from a non-dissolving material such as a non-dissolving biocompatible rigid polymer such as acrylic resin modified polyesters, epoxy resins, UV-curable monomers, resins, silicones.

[061] Therefore, the microneedle 14 provides a sharp needle point, together with enhanced mechanical properties due to the rounded base, the ability to effectively penetrate tissue, a pyramidal tip that serves as a dosage form of bioactive material in which the bioactive material(s) are included in a matrix of dissolving or degradable biomaterial, an undercut core region that enhances mechanical performance during implantation while still providing tissue retention during implantation, and a rapidly dissolving layer that, together with the mechanical mismatch between the dissolving and non-dissolving layers, promotes rapid release of the pyramidal tips.

[062] Microneedle 16 is similar to microneedle 14, but additionally includes a pliable support 34, which may be insoluble. Specifically, the microneedle 16 may be formed with a pyramidal portion that is constructed using a dissolving or biodegradable material, a rapidly dissolving layer may be formed as described above, the remainder of the shaft portion may be made of a non-dissolving material, and the support may be made of a pliable material. , such as an insoluble pliable polymer (silicones, UV-curable polymers, elastomers).

[063] Therefore, the microneedle 16 can provide a sharp needle point, together with enhanced mechanical properties due to the rounded base, the ability to effectively penetrate tissue, a pyramidal tip that serves as a dosage form of the bioactive material in which the bioactive material(s) are included into a matrix of dissolving or degradable biomaterial, and an undercut shaft portion that enhances mechanical performance during insertion while still providing tissue retention during implantation. As in the case of microneedle 14, the rapidly dissolving layer can, together with the mechanical mismatch between the dissolving and non-dissolving layers, promote rapid separation of the pyramidal tips, and the non-dissolving undercut shaft region prevents back diffusion of the embedded bioactive material during both the manufacturing and implantation processes. In this embodiment, the support can adhere more closely to the uneven geometry of the skin surface and, if not dissolved, the support can help prevent moisture absorption during storage, which can cause excessive curvature of the supports and reduce the effectiveness of MNA applications.

[064] FIGS. 2A-2C illustrate the ability of the microneedle array 50, which includes microneedles 52 and a compliant substrate 34, to bend and, after bending, to return to its original state, or at least a state that is closer in shape to the original state than the bent state. .

[065] It should be understood that any combination of features disclosed both in FIG. 1 and throughout this application is intended to be possible. For example, the pliable microneedle substrate 16 and the rapidly dissolving microneedle layer 30 of the microneedles 14, 16 may be used in combination with any of the other designs disclosed herein (eg, microneedles 10, 12).

[066] In addition, the processes disclosed herein can be used to create a microneedle that is formed entirely from insoluble materials for use in the form of coated MNA, tissue adhesive patches (applicators), and/or microprongs. Geometric shapes of a non-dissolving micro-tooth or micro-needle with an undercut have previously been created using additive manufacturing or mechanical micro-machining processes. However, high-throughput fabrication of such geometries has been hampered by the lack of efficient micromolding processes.

[067] Microadditive MNA Manufacturing Technology

[068] In some embodiments, MNAs can be formed using additive manufacturing (AM), including micro-additive manufacturing (µAM) techniques. The methods and systems described herein may allow medical researchers with low microfabrication expertise to directly manufacture their MNA designs from a CAD design without the complex requirements of subtractive manufacturing technology. The μAM methods described herein provide an effective means for the fabrication of novel MNAs designed specifically for effective dermal and non-dermal drug delivery.

[069] In some embodiments disclosed herein, MNA designs include uniquely shaped micrometer-sized needles that contain pointed pyramidal tips and shafts with undercuts and rounded bases to provide effective skin penetration and retention. Unlike conventional MNAs, the MNAs disclosed herein are fabricated via three-dimensional (3D) μAM, assisted by direct 3D laser writing, which offers transformational potential in the field of MNAs, with paralleling levels of simplicity and design capabilities.

[070] As described in detail below, in some embodiments, replicas of the base MNAs can be produced from a mechanically strong, moldable resin in two micromolding steps with high fidelity. These replicas can then be used to prepare production injection molds, such as the polydimethylsiloxane (PDMS) production injection molds that allowed the fabrication of new soluble MNAs with drug-containing tips and undercut microneedles. In some embodiments, the resulting MNAs are fully dissolving MNAs with pronounced undercut features for effective dermal drug delivery.

[071] As shown in FIG. 1, in some embodiments, multiple base MNAs can be printed, replicas of the base MNAs can be quickly obtained, and then multiple replicas can be assembled to create larger MNAs.

[072] In some embodiments, MNAs can combine model antigenic ovalbumin (OVA)±model adjuvant Poly(I:C) from a biosoluble material composition (70%CMC/30%Treh) using a centrifugal casting method. In addition, the proposed MNA designs, together with suitable mold materials, provided a strategic opportunity for direct manufacturing steps without interfering with the molding processes. Fabricated MNAs are particularly effective at penetrating the stratum corneum of the epidermis of living human skin to deliver bioburdens to the dermal microenvironment. These unique MNAs meet the geometric shape and mechanical strength requirements for effective skin penetration for cutaneous vaccination, thereby providing a promising alternative approach for targeted skin immunization techniques.

[073] Exemplary microneedles and arrays and manufacturing systems therefor

[074] FIGS. 3A and 3B depict an exemplary microneedle array (100) (MNA) that includes microneedles with a pointed pyramidal tip (102) and an undercut shaft portion (104). In addition, the microneedles include a rounded base (106).

[075] The methods and systems disclosed herein enable the repeatable production of high quality dissolving MNAs with drug-containing tips and undercut features from various and widely used dissolving microneedle materials such as CMC, PVP (polyvinylpyrrolidone), silk, HA (hyaluronic acid) ), CMC/trehalose, CMC/glucose, CMC/sucrose, PVP/PVA, and any other moldable biosoluble compositions.

[076] In this embodiment, the microneedle has a height (110) that is 50-1500 µm, for example a height of 750 µm, and an angle (A) at the apex of the pyramidal tip that is 10°-60°, for example 30° .

[077] In one embodiment, the microneedle shaft portion may have a width (112) that is 50-500 μm, such as 150 μm. The shaft portion (104) may extend from the bottom edge of the three-dimensional (3D) pyramidal tip to the microneedle substrate (108) with a rounded joint that has a length of 15-75 μm, for example, 35 μm. The width (114) of the bottom edge of the pyramidal tip can be, for example, 100-400 μm, for example, forming a base area of 250 μm x 250 μm.

[078] In some embodiments, the distance (116) between microneedle tips in the array may be 100-800 μm, such as 650 μm. In some embodiments, the MNA may include 1-1000 microneedles, for example, 25 microneedles in a 5 x 5 array configuration on a substrate with an area of 4.75 mm x 4.75 mm.

[079] The rounding at the base (106) of the microneedle helps reduce the associated stress concentrations at sharp corners, which in turn improves the performance of the microneedle during manufacturing and skin insertion processes. The apex angle, width, and height of the microneedles were chosen to provide improved mechanics of insertion into the skin and reduce the number of unsatisfactory results during insertion.

[080] The undercut or anchoring element of the design can optimize retention in the skin during application and, using the new methods and systems disclosed herein, can also be achieved without interfering with processing steps, allowing direct removal of the MNA from injection molds.

[081] The three-dimensional microadditive manufacturing (3D-µAM) approach described here involves creating unique MNA designs from a 3D CAD drawing. In addition, 3D-µAM technology can enable medical researchers with little or no microfabrication expertise to design and fabricate MNA-based drug delivery platforms with application-specific optimizations.

[082] Exemplary manufacturing systems and processes are illustrated in a flowchart in FIG. 4A and graphically depicted in FIG. 4B. As shown in FIGS. 4A and 4B, the process may comprise the following steps to create novel, rapidly dissolving MNAs while achieving high production throughput. In a first step, a 3D CAD drawing (132) of the MNA design can be generated (process step 120). In a second step, direct fabrication of the base MNA (134) from a CAD drawing can be performed via direct 3D laser writing using an insoluble resin (eg, IP-S photoresist) (process step 122). The 3D-µAM technology system could be, for example, the Nanoscribe system (136) for 3D printing.

[083] In the third step, fabrication of a high-precision replica of the base MNA can be rapidly accomplished using a UV-curable resin (eg, VeroWhitePlus, Tangoblack, Digital Materials) and a two-step micromolding method (process step 124). This approach may involve a negative elastomeric injection mold (138) to form a replica (140) of the base MNA.

[084] In a fourth step, original MNA shapes can be created with multiple replicas of the base MNA (eg, six replicas) on 3D printed MNA holders (process step 126). The 3D printed MNA holder (142) may be formed from, for example, a polymer material. Integrating multiple replicas of a base MNA onto, for example, 3D printed MNA holders could allow for larger MNAs and improve productivity.

[085] In a fifth step, production injection molds (144) of MNA from elastomeric PDMS (polydimethylsiloxane) can be made using micromolding (process step 128). In a sixth step, dissolvable MNAs (150) with drug-containing tips can be prepared (process step 130). MNAs may have undercut microneedles incorporating one or more bioactive materials, such as a vaccine or any other bioactive material, made from a water-soluble biocompatible material (eg, compositions of CMC and Treh) by a multi-stage centrifugal casting process.

[086] For example, a bioactive component (e.g., a vaccine) can be centrifugally cast onto the tips of a PDMS production injection mold, and a soluble hydrogel (e.g., CMC/trehalose) can be centrifugally cast into a production injection mold to function as a microneedle construction material and form the MNA substrate . In some embodiments, the base MNA, original MNA molds that include replicas of the base MNA, and elastomeric production injection molds can be reused over a large number of production runs, significantly reducing the cost of manufacturing MNAs with unique designs and increasing productivity.

[087] FIGS. 5A-5E represent final products corresponding to different manufacturing or processing steps. FIG. 5A is an optical microscope image of a base MNA (134) created using direct 3D laser writing, and FIG. 5D is an optical microscope image of an individual undercut needle on a base MNA (134) fabricated by 3D printing. Specifically, the base MNA was fabricated from IP-S photoresist using direct 3D laser writing. IP-S resin is a special material designed for 3D laser lithography and provides high resolution and mechanical integrity of micro- and nanostructures. 3D laser lithography based on two-photon polymerization has provided an effective means to fabricate undercut MNA structures with smooth edges and sharp points, and without any unwanted residues (e.g., machine chips).

[088] FIG. 5B is an optical microscope image of a replica (140) of a base MNA created using a two-step micromolding technique: elastomeric composition molding combined with UV-curing micromolding, and FIG. 5E is an optical microscope image of an individual needle with an undercut on replica (140) of the base MNA. The base MNA was fabricated using a two-step micromolding process. A base IP-S resin MNA was used to fabricate a flexible PDMS injection mold through elastomeric composition molding, and then the PDMS injection mold was used to fabricate multiple VeroWhite resin MNA replicas through UV-curing micromolding. VeroWhite resin is a wear-resistant, acrylic photopolymer that is suitable for use with Polyjet 3D printers.

[089] To enable scalable manufacturing of new MNAs, MNA replicas are then used to create original MNA shapes that include multiple MNAs (eg, six MNA replicas). The final original MNA molds were post-processed in a vacuum oven to facilitate successful curing of the PDMS on the surface of the injection mold, and the PDMS was then fabricated into elastomeric production MNA injection molds that consisted of microneedle-shaped loading wells. FIG. 5C is an optical microscope image of microneedle shaped loading wells in production MNA injection molds (144).

[090] Essentially, these technological steps, together with the greater geometric capabilities of direct laser 3D writing, resulted in a strategy for scalable and efficient MNA fabrication. In addition, the rapid production of 3D printed replicas of the basic MNA using wear-resistant mold material significantly increased productivity. Other undercut features are also possible using other microneedle materials and elastomeric injection molds that are compatible with the processes and systems described herein.

[091] After manufacturing the original MNA molds with six replica MNAs, dissolving MNAs that incorporate the vaccine at the needle tip site were manufactured using a conventional three-step manufacturing strategy from the original mold to the production injection mold to the final dissolving MNAs.

[092] As described in more detail below, dissolvable MNAs that incorporate the vaccine (10 μg OVA ± 25 μg Poly(I:C)) at the undercut needle tip site were manufactured by centrifugal casting technology from two different material compositions (i.e. CMC/trehalose and PVP/PVA). Additionally, CMC/trehalose MNAs with drug-containing tips and undercut microneedles incorporating a colored model drug (eg, doxorubicin) were fabricated to facilitate imaging and demonstrate compatibility with chemotherapeutic agents. The systems and methods described herein have enabled the efficient and rapid production of dissolvable drug-tipped MNAs and undercut microneedles from a variety of dissolvable material compositions.

[093] Figure 6A is an optical microscope image of the final dissolving CMC/Treh-MNAs incorporating bioburdens (eg, OVA+Poly(I:C)). FIG. 6B is an optical microscope image of dissolving PVP/PVA-MNA with drug-containing tips and undercut microneedles incorporating OVA, and FIG. 4C is an optical microscope image of a single CMC/Treh microneedle with drug-containing tip and undercut. including doxorubicin as a colored drug to facilitate visualization.

[094] Example 1 - Fabrication of exemplary MNAs

[095] An exemplary process for creating an MNA is described herein.

[096] Fabrication of basic MNA. The unique MNA design was directly created from the 3D CAD drawing shown in Figure 1 using laser 3D printing (on a Nanoscribe Photonic Professional, GT lithograph) using IP-S photopolymer resist. The Nanoscribe printing system was equipped with a laser generator, an optical communication cabinet, a Zeiss optical microscope attached to a lens to focus the laser beam, a galvanometric mirror system to control the scanning of the laser beam, a piezoelectric stage for precision motion control, and operating software (Nanowrite) to perform 3D printing. print. The entire system was placed on the optical table to dampen vibrations during the printing process.

[097] To fabricate the basic MNA, the MNA design was converted to an "STL" (stereolithography) file format. Next, the STL file was loaded into the specialized software (DeScribe, Germany) of the Nanoscribe system to select the processing mode (i.e. layer acquisition distance, engraving and separation). Finally, the STL file was converted to "GWL" (general record lithography) format to be exported to Nanowrite software for printing the basic MNA. The base MNA was fabricated using galvanometric scanning mode in the XY plane and piezoelectric scanning in the Z direction. The base MNA was divided into 220 μm × 220 μm × 200 μm blocks within the operating range and then composed. The laser power and recording speed were set to 100 mW and 6 cm/s, respectively. The minimum and maximum distances between layers were 0.3 μm and 0.5 μm, respectively. The base MNA was then printed by two-photon polymerization of IP-S photoresist using a 750 nm femtosecond pulsed laser using a unique liquid-immersion mode with a 25x objective and 0.8 numerical aperture in shell and frame mode.

[098] After printing, the base MNA was developed in propylene glycol methyl ether acetate (PGMEA) photoresist solvent for 30 minutes and then washed for 5 minutes in isopropyl alcohol (IPA). After air drying the base MNA, it was irradiated with UV light (365 nm) at an intensity of 16 mW/ ^cm2 for 30 min to further cross-link the part to strengthen the MNA structure.

[099] Making replicas of the base MNA. A two-step micromolding method was performed to produce high-fidelity replicas of the base MNA using a UV-curable resin. First, an elastomeric injection mold, which is the inverse of the base MNA, was made from polydimethylsiloxane (PDMS) using micromolding. Molding of an elastomeric composition using PDMS allows for the precise and reproducible fabrication of replicas with high-precision micrometric-sized structures. Briefly, a base MNA was mounted in a 5 cm diameter petri dish and PDMS was prepared using SYLGARD® 184 two-component curable silicone elastomer (Dow Corning) by mixing base material with curing agent in a 10:1 ratio, SYLGARD® to curing agent. After this, the mixture was poured onto a base MNA placed in a Petri dish and degassed for 15 min. Then, the base MNA with the degassed mixture was placed in an oven and cured at 70°C for 1 hour. The cured PDMS was cooled to room temperature for 5 min and then separated from the base MNA to obtain a PDMS negative injection mold.

[0100] In the second processing step, a negative PDMS injection mold was used to fabricate positive MNA replicas from a UV-curable resin (Stratasys®, VeroWhiteplus-RGD835). For each PDMS injection mold, 20 μL of liquid (uncured) resin was poured onto the injection molds, and then the injection molds were placed in a centrifuge to fill the microneedle mold feed wells with resin at 4500 rpm and 20°C for 1 min. The resin was then cured with UV light (365 nm) at an intensity of 21.7 mW/cm ² for 5 min on each of the top and bottom sides to cure both the base and tips of the microneedles. To ensure the flatness of the base MNA replica substrates, an additional 50 μL of UV-curable resin was poured onto the PDMS injection mold, which was in excess of the remaining available volume. A glass slide was placed on top of the injection mold to remove excess VeroWhite resin, thereby creating a uniform, flat surface on the base. The liquid resin was then cured on the top side for 5 min and then removed from the injection mold to obtain replicas of the base MNA.

[0101] Create original MNA forms. To facilitate scalable fabrication of dissolvable MNAs, original MNA shapes were created by assembling six replicas of the base MNA on MNA holders manufactured by Stratasys® technology from an insoluble photopolymer (VeroWhite) using a high-resolution (16-μm) Polyjet 3D printing system (Objet Connex 500 printer , with a combination of several materials). A 3D model of the MNA holders was created using SolidWorks 2018 CAD software and then converted to an “STL” (stereolithography) file format. Next, specialized software (Objet Studio) cut this 3D model into 2D (2-dimensional) cross layers, creating a computer file that was transferred to the 3D printer system. In the 3D printed MNA holder, channels were designed and fabricated to serve as protruding chambers in production MNA injection molds to serve as support reservoirs for both the bioactive load (e.g., vaccine) and the engineered hydrogel material of the dissolving MNAs in centrifugal casting process. The created original MNA molds were heat treated at 80°C overnight in a vacuum oven to facilitate efficient production of elastomeric production MNA injection molds.

[0102] Making production MNA injection molds. The production MNA injection molds, which included microneedle-shaped loading wells, were made from the commonly used elastomeric polydimethylsiloxane (PDMS) as described for making replicas of the original MNA. The base material was mixed with the curing agent in a ratio of 10:1, SYLGARD® to curing agent. After this, the mixture was poured onto the original MNA mold placed in a Petri dish with a diameter of 10 cm and degassed for 15 minutes. The original mold with the degassed mixture was then placed in a PDMS curing oven at 70°C for 1 hour. After cooling the cured PDMS to room temperature, it was separated from the original MNA mold to produce production PDMS MNA injection molds.

[0103] Production of dissolving MNAs. To prepare dissolvable MNAs with drug-loaded tips and undercut microneedles incorporating OVA±Poly(I:C), a biosoluble materials formulation was used that included carboxymethylcellulose (CMC, catalog no. C5678, Sigma-Aldrich, St. Louis, MO) and trehalose (Treh, catalog no. T9531, Sigma-Aldrich). To prepare the hydrogel form of the dissolving MNA construction material, CMC and Treh powders were thoroughly mixed at a mass ratio of 70% and 30%, respectively. This powder mixture was then added to endotoxin-free water (HyClone HyPure Cell Culture Grade Water) and mixed thoroughly to obtain a concentration of 30% by weight. The prepared hydrogel was cooled at 4°C for 24 hours to bring the mixture to equilibrium. Unique MNA designs with drug-containing tips were then manufactured from CMC/Treh using a multi-stage centrifugal casting method using a centrifuge (Thermo Fisher Scientific Sorvall Legend XTR with Swinging Bucket Rotor TX-750).

[0104] Finally, 5 μl of OVA solution (25 mg/ml OVA in endotoxin-free water) was applied to each of the MNAs on the production PDMS injection molds, and the production injection molds were centrifuged for 1 minute at 20°C and speed of 4500 rpm to fill the microneedle-shaped cavities. After the production injection molds were filled, excess OVA solution was removed from the reservoir. The production injection molds were again centrifuged for 30 min at 20°C and 4500 rpm to ensure that the OVA load was located at the tip region of the microneedle cavities in the production injection molds.

[0105] For MNAs including OVA+Poly(I:C), after loading with OVA, 5 μl of Poly(I:C) solution (62.5 mg/ml Poly(I:C) in endotoxin-free water) applied to each of the MNAs on production PDMS injection molds, and the production injection molds were centrifuged for 1 min at 20°C and 4500 rpm to fill the microneedle-shaped cavities. After the production injection molds were filled, excess Poly(I:C) solution was removed from the reservoir. The production injection molds were again centrifuged for 30 min at 20°C and 4500 rpm to ensure that the Poly(I:C) load was also located on the pyramidal portion of the microneedle cavities in the production injection molds. After placing the bioburdens into the microneedle tips, 40 μg of hydrogel (30% by weight of the 70:30 CMC:Treh formulation) was loaded onto each of the MNAs on production PDMS injection molds to fill the geometric shapes of the microneedles in the production injection molds and form the MNA substrates. The hydrogel-filled production injection molds were then centrifuged for 5 h at 20°C and 4500 rpm to obtain dissolving MNAs containing 10 μg OVA ± 25 μg Poly(I:C). Due to the special geometry of the MNA production injection molds, each replica manufacturing operation simultaneously produced six matrices in the form of dissolving MNAs containing OVA±Poly(I:C).

[0106] To demonstrate the fabrication of dissolvable MNAs containing undercut microneedles from other water-soluble material (i.e., possible materials), a biosoluble polymer composition of polyvinylpyrrolidone (PVP) and polyvinyl alcohol (PVA) was also used to fabricate MNAs with different bioburdens. (40% by weight of 60:40 PVP:PVA composition). To evaluate the geometric integrity of the new undercut microneedles, bright-field optical microscopy images were obtained of fabricated base MNAs, replica MNAs, elastomeric production MNA injection molds, and dissolving MNAs containing OVA± Poly(I:C).

[0107] Dermal delivery using exemplary MNAs

[0108] Preparation of ex vivo human skin explants. Human skin explants were prepared from deidentified healthy plastic surgery donors, obtained through the Pitt Biospecimen Core facility, and used in accordance with the University of Pittsburgh Medical Center guidelines. The tissue was washed in 70% ethanol and then in phosphate-buffered saline (PBS). Human skin explants (approximately 1 mm thick) were harvested for examination using a miniature Silver skin grafting knife (Padgett, Integra Miltex, Plainsboro, NJ) and then cut into 20 mm × 20 mm square flaps. The resulting human skin samples consisted of intact epidermis and a thin layer of underlying dermis and were maintained as explants in a normal physiological state by air-fluid interface culture.

[0109] Image analysis. To evaluate the intradermal delivery of bioactive materials (eg, vaccines) from MNA into living human skin explants, several image analyzes were performed. Using the above procedure, CMC/Treh-MNA was prepared with drug-loaded tips incorporating a dyed load (ie, Allura Red R40). Before using MNA on human skin explants, MNA images were obtained using a bright-field optical microscopy technique.

[0110] MNA was then applied to human skin explants and removed after 10 minutes. Thereafter, target areas of human skin were examined under bright-field microscopes to obtain images of deposition patterns of the stained CMC/Treh-MNA bioburden into human skin. Materials remaining in the MNA were also visualized by optical microscopy after application to human skin. To further quantify intradermal MNA vaccine delivery into human skin, CMC/Treh-MNAs were prepared that included both Alexa555-labeled OVA and Alexa488-labeled Poly(I:C) and incorporated these matrices into human skin at 10 minutes and removed. The human skin explants were then analyzed histologically. Briefly, human skin samples treated with MNA were fixed in 2% paraformaldehyde and then soaked in sucrose solution, with 3 changes of this solution, for 24 hours. Tissue sections were then quickly frozen in histological compound at optimal cutting temperature (OCT). and cryosectioned into sections approximately 10 µm thick. Sectioned human skin samples were counterstained with the nuclear fluorescent dye DAPI (4',6-diamidino-2-phenylindole). The stained sections were then imaged using a Nikon fluorescence transmission microscope to detect OVA with Alexa555 and Poly(I:C) with Alexa488 in cross sections of human skin, and also with a bright field microscope to better show the passage through the stratum corneum of the epidermis.

[0111] In vivo cutaneous immunization with MNA

[0112] Keeping mice and animals. Female C57BL/6J mice were purchased from Jackson Laboratory (Bar Harbor, Maine) and used at 8–10 weeks of age. Mice were maintained under specific pathogen-free conditions at the University of Pittsburgh, and all experiments were performed in accordance with Institutional Animal Care and Use Committee (IACUC) guidelines.

[0113] In vivo imaging using IVIS technology. In vivo intradermal vaccine delivery using novel dissolving MNAs was demonstrated in the C57BL/6J mouse. CMC/Treh-MNA with drug-containing tips, including both OVA labeled with Alexa555 and Poly(I:C) labeled with Alexa488, were prepared using the technology described above and applied to the abdomen of a mouse that was temporarily anesthetized by inhalation of fluorinated ether within 10 min. Next, the MNA was removed and the mouse was returned to its normal active state. Images of MNAs containing OVA+Poly(I:C) were obtained before and after in vivo application. Images of mice treated with MNA were acquired using IVIS 200 in vivo imaging systems (PerkinElmer), using filters to analyze for fluorescently labeled Poly(I:C) and OVA at the site of MNA application. The image was post-processed using Living Image software (PerkinElmer).

[0114] Cell-mediated and humoral immune responses. To demonstrate skin vaccination with the novel MNAs, with drug-loaded tips, CMC/Treh-MNAs incorporating 10 μg OVA ± 25 μg Poly(I:C) were prepared as described above. Mice were immunized by skin vaccination with MNA (10 μg OVA ± 25 μg Poly(I:C), with MNA applied to the right and left sides of the abdomen, two MNAs per mouse), or by two intramuscular injections of 10 μg OVA in solution PBS was injected into the gastrocnemius muscle of the hind limb or left without treatment (i.e., not used in the experiments). Immunizations were performed on days 0 and 7 as primary and additional doses, respectively. Mice were then analyzed for in vivo OVA-specific T-cell cytotoxic activity and OVA-specific humoral immune response 5 days after supplemental dosing using conventional methods.

[0115] To analyze the OVA-specific humoral immune response, anesthetized mice were bled at the time of sacrifice by cardiac puncture and serum was isolated using a BD Microtainer serum separator (BD Biosciences, San Jose, Calif.). Serum OVA-specific IgG1 and IgG2c antibodies were measured by indirect enzyme-linked immunosorbent assays (ELISA). Costar EIA/RIA plates (Corning Inc., Corning, NY) were coated with OVA (100 μg/ml in 0.5 M carbonate-bicarbonate buffer, pH 9.6 ; Sigma) by overnight incubation at 4°C. The plates were washed (3×) with 0.05% Tween20 in PBS and sealed with 1% goat serum in PBS for 1 hour at 37°C. Serum and standard samples (anti-OVA IgG1 from Cayman Chemical, Ann Arbor, MI; anti-OVA IgG2c from Chondrex, Redmond, WA) were diluted in 1% goat serum, added to the plates, and incubated for 2 hours at 37°C. . After washing (3×), plates were incubated for 1 hour at 37°C with biotinylated secondary antibodies (goat anti-mouse IgG1 or IgG2c, 1:20,000 in 1% goat serum; Jackson ImmunoResearch, West Grove, PA ). The plates were then washed (3×) and incubated for 30 min with streptavidin-HRP (1:1000 in 1% goat serum; BD Biosciences). The plates were washed again (3x) and incubated at room temperature with 4,4',5,5'-tetramethylbenzidine (TMB) peroxidase substrate (Sigma) for 2-3 minutes, and the reaction was quenched with 1.0-M solution H2SO4. For all ELISAs, absorbance at 450 nm (OD450) was measured using a SpectraMax 340PC plate reader (Molecular Devices, Sunnyvale, CA), and serum concentrations were calculated from standard curves.

[0116] To assess OVA-specific cytotoxic activity of T cells, splenocytes from mice not used in the experiments were sensitized with a solution of 2 μg/ml OVA257-264 peptide (SIINFEKL) or left unsensitized for 1 hour. Antigen-sensitized splenocytes were washed and stained with high-concentration carboxyfluorescein succinimidyl ester (CFSE, 10 μM), and nonsensitized splenocytes were labeled with low-concentration CFSE (1 μM) for 15 min at 37°C. A 1:1 mixture of sensitized target cells and non-sensitized control cells (107 each) was injected intravenously (IV) into immunized mice and mice not used in the experiments. Twenty-four hours after adoptive transfer, mouse spleens were removed and target cell lysis was assessed by comparing antigen-sensitized and non-sensitized populations by flow cytometry to quantify OVA-specific lysis of SIINFEKL-sensitized target cells labeled with a high-concentration CFSE solution. Specific lysis was calculated and expressed as a percentage of maximum lysis as % Lysis = {1 − [(average CFSElow/CFSEhigh ratio in unvaccinated mice/(CFSElow/CFSEhigh ratio in vaccinated mouse)]} × 100.

[0117] Intradermal and non-dermal applications

[0118] In addition to skin vaccination, the MNAs described herein can be used in a wide range of applications for intradermal and nondermal (eg, ocular and cardiac tissue) drug delivery. As shown in FIGS. 7A-D, the bioburden of interest can be placed at the very tip of the microneedles during the centrifugal casting process (ie, the pyramid region), or the entire pyramidal region can be filled with bioburden depending on dose requirements. FIG. 7A shows PVP/PVA-MNA containing dextran (molecular weight about 40 kDa) stained with Texas Red on microneedle tips, and FIG. 7B shows CMC/Treh-MNA containing Allura Red R40 dye (molecular weight about 500 kDa) in the pyramidal region of the microneedles.

[0119] Additionally, multiple repeatable centrifugal casting steps can be performed to produce high-quality MNAs with undercut microneedles that incorporate multiple loads in their pyramidal regions (FIGS. 7C and 7D). FIG. 7C represents PVP/PVA-MNA with drug-containing tips including multiple loads, e.g., Texas Red-stained dextran and Allura Red R40, and FIG. 7D represents PVP/PVA-MNA with drug-containing tips including multiple loads. , such as Texas Red-stained dextran and Alexa488-labeled PLGA microparticles (average diameter 10 μm).

[0120] As such, the proposed subsurface and novel MNA designs are compatible with mono- and combination therapies in several dermal and non-dermal applications. It is important to note that the same production injection molds can be used to efficiently produce dissolving MNAs with undercut microneedles over multiple cycles (e.g., Figures 7A and 7C depict MNAs produced after the first and twelfth cycles, respectively, using the same technology ). Importantly, the present study demonstrates the applications of single-load (OVA) and multiple-load (OVA+Poly(I:C)) MNAs for in vivo skin vaccination.

[0121] Additive manufacturing technology, or 3D printing, used in the systems and methods described in this application allows for the accurate and repeatable production of complex 3D geometric shapes, without design restrictions, and offers a high degree of flexibility and design control. By using the systems and methods described herein, it is possible to reduce design to manufacturing time for optimal custom drug delivery systems.

[0122] Various geometric shapes of needles

[0123] As described above, the systems and methods disclosed herein offer an unprecedented level of design flexibility for MNA structures. To demonstrate the range of geometric capabilities of direct laser 3D writing, microneedle structures with a variety of geometric shapes were fabricated.

[0124] FIGS. 8A-8B show that this technology can produce microneedles with geometric shapes in a wide range and with high precision, in order to optimize according to the application. Additionally, as shown in FIGS. 8A-8B, it allows designs to be varied over a wide range, including height, width, apex angle, and microneedle geometry, without the need for complex and special processing steps. As such, this technology paves the way for the design and manufacture of unique MNA structures for specialized applications.

[0125] Intradermal delivery of MNA vaccine to human skin.

[0126] To evaluate the dermal delivery characteristics of bioburdens from MNAs with undercut needles, MNAs with drug-containing tips and Allura Red R40 dye were fabricated using the presented fabrication methods. Human skin explants were prepared as described above. MNAs containing Allura Red R40 dye were applied to living human skin explants and removed after 10 min. Images of these MNAs before (Figure 9A) and after (Figure 9B) application demonstrated high MNA quality and complete dissolution of the microneedles, respectively. Corresponding MNA-embedded load deposits (eg, Allura Red R40) in the target skin are shown in Figure 9C.

[0127] Successful delivery of the vaccine through the stratum corneum of the epidermis into the skin microenvironment with immune cells is of great importance for effective intradermal immunization. To anatomically assess the delivery of OVA and Poly(I:C) to human skin, CMC/Treh-MNA containing both Alexa555-labeled OVA and Alexa488-labeled Poly(I:C) were applied to human skin explants. for 10 minutes and then removed. Target human skin was cryosectioned and imaged using a Nikon fluorescence transmission microscope. Histology demonstrated microneedles penetrating through the epidermis into the dermis (Figure 9G) and delivery of fluorescently labeled OVA and Poly(I:C) to targeted human skin microenvironments (Figure 9D-I: DAPI nuclear stain, Poly(I:C) , tagged with Alexa488, OVA tagged with Alexa555, brightfield, overlay of 3 different fluorescent colors, merged image of all fluorescent images with brightfield image, respectively). In Fig. 9D-I, scale bars correspond to 100 μm.

[0128] Together, these images suggest that the proposed unique MNAs met the geometry (i.e., sharp points and smooth edges) and mechanical strength requirements for safe penetration of human skin (i.e., passage through the stratum corneum and living epidermis epidermis) and material requirements for effective dissolution in the aqueous environment of the skin, and therefore provide a platform for effective dermal delivery of drugs and vaccines.

[0129] In vivo cutaneous immunization from MNA

[0130] To study in vivo intradermal vaccine delivery in mice using the novel MNAs, CMC/Treh-MNAs with precision undercut microneedles containing both Alexa555-labeled OVA and Poly(I:C) were fabricated as described above ), tagged with Alexa488. Before use, MNAs containing OVA with Alexa555+Poly(I:C) were imaged by bright-field optical microscopy and epifluorescence microscopy (Figure 10A). MNA was then applied to the mouse and removed after 10 min. Using bright field optical microscopy, the remaining material in the MNA after overlays was also imaged (FIG. 10B). Images of MNA-treated mice were acquired using an IVIS 200 live animal imaging system, with filters for both Poly(I:C) with Alexa488 and OVA with Alexa555. Co-delivery of Poly(I:C) and OVA from MNA is shown in Figures 10C and 10D, respectively. Taken together, the above images demonstrated the successful in vivo application of the novel MNAs in mice and, in turn, the effective cutaneous delivery of MNA drugs and vaccines using the novel MNA constructs.

[0131] Following the demonstration of successful intradermal delivery of vaccines in vivo, we specifically assessed the immunogenicity of the antigen±adjuvant contained in MNA and compared immunization using MNA with vaccination using the clinically common intramuscular (IM) injection method. To this end, CMC/Treh-MNAs were prepared with 10 μg OVA ± 25 μg Poly(I:C) in each MNA as described above. The inventors and other researchers had previously shown that soluble proteins bundled into MNAs maintained their integrity. For immunization, the vaccination regimen described in the methods section was performed to immunize mice both intramuscularly and from MNA via abdominal sites. OVA-specific cytotoxic T cell (CTL) and antibody responses were quantified using a standard in vivo lytic assay and ELISA, respectively. Skin vaccination with MNAs induced robust antigen-specific cellular immune responses (FIG. 11A-B). As expected, equivalent amounts of antigen-sensitized (CFSE high) target cells and non-sensitized (CFSE low) target cells were recovered from naïve or nonimmunized mice (Figure 11A), indicating the absence of antigen-specific cytolytic activity. In contrast, specific lysis of antigen-sensitized target cells was dramatically enhanced in immunized mice, as shown by the reduced survival of OVA-sensitized targets compared with nonsensitized targets. Specifically, while intramuscularly OVA-immunized mice showed minimal differences between the secretions of the two populations (Fig. 11A), mice immunized with MNA OVA exhibited greater OVA-specific lysis in vivo, with significantly less intramuscular injection of OVA) showed less survival and release of OVA-sensitized targets than non-sensitized targets (Fig. 11A). Immunization with OVA+Poly(I:C) further increased vaccination efficiency (Fig. 11A). Quantitative determination of antigen-specific lysis by standard methods confirmed that immunization with MNA induced strong CTL (cytotoxic T cell) immunity (Fig. 11B), which was further enhanced by the inclusion of Poly(I:C) adjuvant. Intramuscular immunization produced weaker responses compared to unimmunized controls. In combination, the above results showed that these MNAs can effectively deliver antigens ± adjuvants into microenvironments with APCs (antigen presenting cells) inside the skin to induce strong immune responses generated by CTL.

[0132] In addition to the immunity provided by CTL, cutaneous vaccination with MNA induced robust antigen-specific humoral immune responses (FIG. 11C). Although vaccination of mice with both MNA and intramuscular injections resulted in IgG production, mice vaccinated with dissolving MNAs had significantly higher IgG titers, demonstrating the importance of MNA technology for immunization efforts. Finally, the presented new MNAs provide a unique opportunity for the specific and precise delivery of encapsulated antigen±adjuvant to defined microenvironments within the skin. For example, the inventors and others have shown that the skin contains large numbers of dendritic cells and other antigen-presenting cells (APCs) that determine vaccine stimulation of the immune system. Therefore, targeting cutaneous APCs with MNA may be an effective strategy for vaccination in general and in particular for inducing cell-mediated immune responses, including cytotoxic T cell (CTL) immune responses, important for the prevention or treatment of many infectious diseases and cancer. As such, the proposed MNA technology could provide capabilities suitable for multiple vaccination strategies.

[0133] Accordingly, the development and use of new dissolving MNAs with undercut microneedles provide effective intradermal vaccination. Unique MNA designs include pyramidal tips and bar undercut areas with rounded bases. The fabrication method for creating undercut MNAs fundamentally involved laser 3D printing and a series of micromolding processes. Successful and reproducible fabrication of soluble MNAs with undercut needles that incorporate a large number of bioburdens has been achieved using a variety of biocompatible and water-soluble polymers. Importantly, an unprecedented level of geometric capabilities of laser 3D printing has been demonstrated to demonstrate purpose-built MNA designs for several dermal and non-dermal drug delivery applications. The proposed novel MNAs with undercut features met the strength requirements for safe penetration into the skin of humans and mice and successfully delivered bioburdens to target skin microenvironments. Importantly, cutaneous vaccination using model antigen-loaded MNAs (OVA-MNAs) elicited stronger antigen-specific cellular and humoral immune responses than vaccinations obtained by the traditional intramuscular injection vaccination method (IM-OVA). Most importantly, co-delivery of antigen (OVA) with adjuvant (Poly(I:C)) from MNA enhanced vaccine immunogenicity compared with intramuscular vaccination, indicating increased efficacy of cutaneous vaccination with MNA. Overall, the presented approach provides an efficient means of fabricating novel dissolving MNAs for a wide range of dermal and nondermal drug delivery applications.

[0134] In view of the numerous possible embodiments to which the principles of the disclosed invention can be applied, it should be recognized that the illustrated embodiments are only preferred examples of the invention and should not be considered limiting the scope of the invention. On the contrary, the scope of the invention is determined by the following claims. Therefore, the inventors claim by their invention everything that falls within the scope and spirit of this claim.

Claims (79)

1. A method for forming a microneedle matrix, comprising the steps of: forming a production injection mold, wherein the production injection mold contains cavities that are shaped to define respective microneedles, each of which has a shaft, a microneedle tip, a rounded base and at least one undercut element, include at least one bioactive material in the first soluble material to provide a biodegradable matrix, delivering the biodegradable matrix to at least a portion of the microneedle tip defined by corresponding cavities of the production injection mold; form microneedles in a production injection mold, which contain a biodegradable matrix; And removing the microneedles from the production injection mold by pulling the microneedles from the injection mold, wherein the production injection mold is formed from a material that has sufficient elasticity to allow the molded microneedle array to be removed from the production injection mold in a single pull without damaging the integrity of the microneedle shape defined by the injection mold. 2. The method according to claim 1, wherein said at least one undercut element is located directly below the tip of the microneedle. 3. The method according to claim 1 or 2, wherein the rod is formed from the first soluble material. 4. The method according to claim 1 or 2, in which the rod is formed from an insoluble material. 5. The method according to any one of claims 1 to 4, wherein forming the microneedle arrays comprises supplying a second soluble material to the production injection mold to form a soluble layer on a portion of the shaft that is adjacent to the tip of the microneedle. 6. The method of claim 5, wherein the second soluble material of the dissolving layer is selected from a material that dissolves faster than the biodegradable or biosoluble matrix that contains the bioactive material. 7. The method according to any one of claims 1 to 6, further comprising the step of forming a substrate near the lower portion of the rod. 8. The method according to claim 7, wherein the substrate is formed from a soluble material. 9. The method according to claim 7, wherein the substrate is formed from an insoluble material. 10. The method of claim 7, wherein the backing is formed from a material that is bendable, deformable, or flexible to conform tightly to the uneven geometric shape of the skin surface. 11. The method according to any one of claims 1 to 10, wherein the first soluble material contains carboxymethylcellulose, trehalose, polyvinylpyrrolidone, maltodextrin, silk, hyaluronic acid, polyvinyl alcohol, polyethylene glycol, copolymer of lactic acid-glycolic acid, polylactic acid, or a combination thereof. 12. The method of claim 5, wherein the second soluble material comprises a rapidly dissolving low molecular weight polymer. 13. The method of claim 12, wherein the second soluble material is glucose, trehalose, sucrose, maltodextrin, polyvinylpyrrolidone, or a combination thereof. 14. The method of claim 10, wherein said material is an insoluble polymer. 15. The method according to any one of claims 1 to 14, wherein said at least one bioactive material contains two or more bioactive materials. 16. The method according to any one of claims 1 to 15, wherein said one or more bioactive materials contain cosmetics, dermal fillers, statins, growth factors, analgesics, antihistamines, vitamins, anesthetics, anti-aging agents, small molecule drugs, haptens, allergens, anti-inflammatory agents, proteins, peptides, microbubbles, exosomes, polyplexes (small interfering RNA, short hairpin RNA, DNA vector complexes), recombinant viral vectors (i.e. adenovirus, lentivirus, vaccinia virus, adeno-associated virus, and their different serotypes), monoclonal and polyclonal antibodies and living or lysed cells. 17. The method according to any one of claims 1 to 16, wherein the microneedle has a shape that includes a round needle, a square needle, a beveled needle, a pyramid on a square shaft, an arrowhead on a shaft, a pyramidal tip on a square needle, a conical tip on a round needle rod, square needle, 45° pyramid or 30° pyramid. 18. The method according to any one of claims 1 to 17, wherein the microneedle further comprises a rounded portion at the intersection of the substrate and the rod. 19. Microneedle matrix formed by the method according to any one of claims 1 to 18. 20. A microneedle array formed using a one-pull ejection mold, comprising: substrate; microneedles; And at least one bioactive material combined with the first soluble material to form a biodegradable matrix, wherein the microneedles each contain a rod, a microneedle tip, a rounded base and at least one undercut element. 21. Microneedle matrix according to claim 20, in which the undercut element is located at the intersection of the rod and the tip of the microneedle. 22. The microneedle array of claim 20 or 21, wherein the core is formed from a first soluble material. 23. Microneedle array according to claim 20 or 21, wherein the rod is formed from an insoluble material. 24. The microneedle array according to any one of claims 20 to 23, wherein the microneedles each contain a second soluble material that forms a rapidly dissolving layer on a portion of the shaft that is adjacent to the tip of the microneedle. 25. The microneedle matrix of claim 24, wherein the second soluble material of the dissolving layer is selected from a material that dissolves faster than the biodegradable matrix that contains the bioactive material. 26. Microneedle matrix according to claims 20-25, in which the substrate is formed from a soluble material. 27. Microneedle matrix according to claims 20-25, in which the substrate is formed from an insoluble material. 28. The microneedle array of claims 20 to 27, wherein the backing is formed from a material that is flexible to conform to the uneven geometric shape of the skin surface. 29. The microneedle matrix of claims 20 to 28, wherein the first soluble material contains carboxymethylcellulose, trehalose, polyvinylpyrrolidone, maltodextrin, lye, hyaluronic acid, lactic acid-glycolic acid copolymer, polylactic acid, polyvinyl alcohol, polyethylene glycol, or a combination thereof. 30. The microneedle array of claim 24 or 25, wherein the second soluble material comprises a rapidly dissolving low molecular weight polymer. 31. The microneedle array of claim 30, wherein the second soluble material is glucose, trehalose, sucrose, polyvinylpyrrolidone, maltodextrin, or a combination thereof. 32. The microneedle array of claim 28, wherein said material is an insoluble polymer. 33. Microneedle array according to claims 20-32, wherein said at least one bioactive material contains two or more bioactive materials. 34. Microneedle matrix according to claims 20-33, wherein said one or more bioactive materials contain cosmetics, dermal fillers, statins, growth factors, analgesics, antihistamines, vitamins, anesthetics, anti-aging agents, small molecule drugs, haptens , allergens, anti-inflammatory agent, proteins, peptides, microbubbles, exosomes, polyplexes (small interfering RNA, short hairpin RNA, DNA vector complexes), recombinant viral vectors (i.e. adenovirus, lentivirus, herpes simplex virus, vaccinia virus, adeno-associated virus and their different serotypes), monoclonal and polyclonal antibodies and live or lysed cells. 35. The microneedle array of claims 20 to 34, wherein the microneedle has a shape that includes a round needle, a square needle, a beveled needle, a pyramid on a square shaft, an arrowhead on a shaft, a pyramidal tip on a square needle, a conical tip on round needle rod, square needle, 45° pyramid or 30° pyramid. 36. Microneedle matrix according to claims 20-35, in which the microneedle additionally contains a rounded section at the intersection of the substrate and the rod. 37. A method for forming an injection mold, containing the following steps: creating a 3D CAD drawing of a microneedle array that contains microneedles with at least one undercut element; form a basic microneedle matrix using a 3D CAD drawing; forming at least one replica of the basic microneedle array; And forming a production injection mold of the microneedle matrix using said at least one replica, wherein the production injection mold is formed from a material that has sufficient elasticity to allow the molded microneedle array to be removed from the production injection mold in a single pull without damaging the integrity of the microneedle shape defined by the injection mold. 38. The method of claim 37, wherein said at least one replica includes replicas, and forming the production injection mold comprises forming a microneedle die holder and combining the replicas on the microneedle die holder. 39. The method according to claim 37 or 38, wherein the microneedle array holder is formed from a polymeric material. 40. A method for forming an adhesive applicator for body tissue, comprising the steps of: forming a production injection mold, wherein the production injection mold contains cavities that are shaped to define respective microneedles, each of which has a shaft, a microneedle tip, a rounded base and at least one undercut element, include at least one bioactive material in the first soluble material to provide a biodegradable matrix, delivering the biodegradable matrix to at least a portion of the microneedle tip defined by corresponding cavities of the production injection mold; form microneedles in a production injection mold, which contain a biodegradable matrix; And removing the microneedles from the production injection mold by pulling the microneedles from the injection mold, wherein the production injection mold is formed from a material that has sufficient elasticity to allow the molded microneedle array to be removed from the production injection mold in a single pull without damaging the integrity of the microneedle shape defined by the injection mold. 41. A method for forming an adhesive applicator for body tissue or an array of micro-teeth, comprising the steps of: forming a production injection mold, wherein the production injection mold contains cavities that are shaped to define respective microneedles, each of which has a shaft, a microneedle tip, a rounded base and at least one undercut element, include at least one bioactive material in the first soluble material to provide a biodegradable matrix, delivering the biodegradable matrix to at least a portion of the microneedle tip defined by corresponding cavities of the production injection mold; form microneedles in a production injection mold, which contain a biodegradable matrix; And removing the microneedles from the production injection mold by pulling the microneedles from the injection mold, wherein the production injection mold is formed from a material that has sufficient elasticity to allow the molded microneedle array to be removed from the production injection mold in a single pull without damaging the integrity of the microneedle shape defined by the injection mold. 42. A method for forming a microneedle matrix, comprising the steps of: forming a production injection mold, wherein the production injection mold contains cavities that are shaped to define respective microneedles, each of which has a shaft, a microneedle tip, a rounded base and at least one undercut element, form microneedles in a production injection mold; removing the microneedles from the production injection mold by pulling the microneedles from the injection mold; And covering at least a portion of the microneedles with a bioactive component, wherein the production injection mold is formed from a material that has sufficient elasticity to allow the molded microneedle array to be removed from the production injection mold in a single pull without damaging the integrity of the microneedle shape defined by the injection mold. 43. The method of claim 42, wherein the microneedle is coated with the bioactive component by dip coating, compressed air spraying, or a combination thereof. 44. A method of using any of the microneedle matrices formed according to one of claims 20-36, the method comprising the step of: a microneedle matrix is applied to the target area of the skin for intradermal delivery of at least one bioactive component. 45. A method of using any of the microneedle matrices formed according to one of claims 20-36, the method comprising the step of: applying a microneedle array to the non-skin tissue to deliver at least one bioactive component to the non-skin tissue.