Microneedle Arrangements with Relief Features for Cutaneous and Non-Cutaneous Drug Administration, Methods for Forming Microneedle Arrays and Molds

Abstract

Microneedle arrangements with embossed features for cutaneous and non-cutaneous drug administration. The disclosed methods relate to the formation of microneedle arrangements from a production mold comprising a flexible mold material. The production mold includes a plurality of cavities that are shaped to define a plurality of respective microneedles, each possessing a shaft, a microneedle tip, a threaded base, and at least one embossed feature. The flexible material possesses sufficient elasticity to allow the molded microneedle arrangement to be removed from the production mold in a single pull without damaging the integrity of the microneedle shape as defined by the mold, as well as retaining the integrity of the mold for multiple uses. The microneedles may possess a variety of geometric structures and may include different dissolvable and insoluble layers, as well as different types of support layers.

Description

1 / 39 “MICRONEEDLE ARRANGEMENTS WITH ELEVATION CHARACTERISTICS FOR CUTANEOUS AND NON-CUTANEOUS DRUG ADMINISTRATION, METHODS OF FORMING MICRONEEDLE ARRANGEMENTS AND MOLD” CROSS-REFERENCE ON RELATED REQUEST

[001] This application claims the benefit for the Patent Application Provisional US No. 62 / 848,939 filed on May 16, 2019, which is incorporated herein by reference in its entirety. FIELD

[002] This disclosure is directed to microneedle arrays and, in particular, to microneedle arrays formed with relief features for cutaneous and non-cutaneous drug delivery, as well as for tissue adhesives. ACKNOWLEDGMENT OF GOVERNMENTAL SUPPORT

[003] This invention was developed with government support under grant numbers AR071277 and AR074285 granted by the National Institutes of Health. The government owns certain rights to the invention. BACKGROUND

[004] The skin is an easily accessible tissue with several distinct functions, such as serving as a protective barrier and a thermal regulator. Notably, the skin also functions as an active immune organ. For some purposes, such as immunization, cancer immunotherapy, and allergy desensitization, the skin may be the preferred anatomical target site, as it harbors large populations of professional antigen-presenting cells and immune accessory cells. Most vaccines, immunomodulators, and cancer drugs are administered using needle-based hypodermic injections. However, the administration of drugs using traditional syringes is Petition 870260056666, dated 11 / 06 / 2026, page 9 / 131 2 / 39 associated with several disadvantages. These include the need for trained healthcare personnel for administration, trypanophobia (i.e., fear of needles), low patient cooperation, risk of disease transmission and needle stick injuries, and the costs of cold chain storage and transport.

[005] Furthermore, parenteral injections fail to deliver bioburdens reproducibly and accurately to targeted skin microenvironments. Thus, vaccines and drugs administered by conventional injections may result in suboptimal efficacy. Collectively, these factors hinder the effective use of vaccines, cancer drugs, or immunomodulators and lead to ineffective cutaneous immunization and treatment strategies. SUMMARY

[006] Various methods and systems are disclosed in this document in connection with the manufacture of microneedle arrays and, in particular, microneedle arrays formed with relief features for cutaneous and non-cutaneous drug delivery, as well as for tissue adhesives.

[007] In some of the embodiments described below, a method for forming a microneedle arrangement comprises forming a production mold of a flexible material having a plurality of cavities that are molded to define a plurality of respective microneedles, each of which has a shaft, a microneedle tip, a filament base and at least one relief feature, incorporating at least one bioactive material in a first dissolvable material to provide a biodegradable matrix, distributing the biodegradable matrix at least in the part of the microneedle tip defined by the respective cavities of the production mold, forming a plurality of microneedles in the production mold that include the biodegradable matrix, and removing the Petition 870260056666, dated 11 / 06 / 2026, page 10 / 131 3 / 39 microneedles are removed from the production mold by pulling the microneedles out of the mold. The flexible material must possess sufficient elasticity to allow the molded microneedle arrangement to be removed from the production mold in a single pull, without damaging the integrity of the microneedle shape as defined by the mold.

[008] In other embodiments, a microneedle array is formed using a single pull mold comprising a support layer, a plurality of microneedles, and at least one bioactive material combined with a dissolvable first material to form a biodegradable matrix. Each of the plurality of microneedles has a shaft, a microneedle tip, a filament base, and at least one relief feature.

[009] In still other embodiments, a method of forming a mold comprises generating a 3D-CAD drawing of a microneedle arrangement that includes a plurality of microneedles with at least one relief feature, forming a master microneedle arrangement using the 3D-CAD drawing, forming at least one replica of the master microneedle arrangement, and forming a production mold of the microneedle arrangement using at least one replica. The production mold is made of a flexible material.

[0010] The above and other objectives, features and advantages of the invention will become more apparent from the following detailed description, which continues with reference to the accompanying figures. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] FIG. 1 illustrates illustrative microneedle designs that, in these examples, have sharp-tipped conical heads, circular shafts with relief, and threaded bases.

[0012] Figures 2A to 2C illustrate a microneedle arrangement that Petition 870260056666, dated 11 / 06 / 2026, page 11 / 131 4 / 39 is formed with an adaptable support layer.

[0013] FIG. 3A shows a computer-aided design (CAD) drawing of an example of MNAs that include sharp needles with relief features and fillet bases.

[0014] FIG. 3B shows an illustrative 3D CAD drawing of a MNA with a 5x5 needle arrangement.

[0015] FIG. 4A shows an illustrative flowchart for an MNA manufacturing process.

[0016] FIG. 4B illustrates the different stages of the MNA manufacturing process illustrated in FIG. 4A.

[0017] FIG. 5A shows an optical microscope image of a master MNA created using direct 3D laser engraving.

[0018] FIG. 5B shows an optical microscope image of the master MNA replica created using a two-step micromolding strategy.

[0019] FIG. 5C shows an optical microscope image of the microneedle-shaped wells in MNA production molds.

[0020] FIG. 5D shows an optical microscope image of an individually embossed needle in the 3D printed master MNA.

[0021] FIG. 5E shows an optical microscope image of a needle with individual relief in the replica of a master MNA.

[0022] FIG. 6A shows an optical microscope image of End-dissolving MNAs (CMC / Treh) incorporating bioburden (e.g., OVA + Poly(I:C)).

[0023] FIG. 6B shows an optical microscope image of MNAs PVP / PVA with dissolution-loaded tips featuring raised microneedles integrating OVA.

[0024] FIG. 6C shows an optical microscope image of a single CMC / Treh microneedle with loaded tips. Petition 870260056666, dated 11 / 06 / 2026, page 12 / 131 5 / 39 incorporating Doxorubicin as a colored drug to facilitate image representation.

[0025] FIG. 7A shows PVP / PVA MNAs incorporating Dextran labeled with Texas Red (molecular weight of approximately 40 kDa) on the tips of the microneedles.

[0026] FIG. 7B shows CMC / Treh microneedles with a loaded tip incorporating Allura Red R40 dye (approximate molecular weight of 500 Da) in the pyramid region of the microneedles.

[0027] FIG. 7C shows PVP / PVA MNAs with filled tips incorporating various fillers, such as Dextran labeled Texas Red and Allura Red R40 dye.

[0028] FIG. 7D shows PVP / PVA MNAs with loaded tips incorporating multiple fillers, such as Dextran labeled Texas Red and PLGA microparticles labeled Alexa488 (average diameter of 10 µm).

[0029] FIG. 8A illustrates different microneedle designs manufactured from photoresistant IP-S using 3D direct laser engraving.

[0030] FIG. 8B illustrates images of microneedles printed with filament bases.

[0031] FIG. 9A illustrates optical microscope images of MNAS PVP / PVA incorporating Allura Red dye before application to human skin.

[0032] FIG. 9B shows a bright-field microscope image of microneedle traces of Allura Red R40 dye in samples of live human skin.

[0033] FIG. 9C shows optical microscope images of PVP / PVA-MNAs incorporating Allura Red dye after application to human skin.

[0034] FIGS. 9D to 9I show intradermal co-administration Petition 870260056666, dated 11 / 06 / 2026, page 13 / 131 6 / 39 Poly (I:C) labeled Alexa488 and OVA labeled Alexa555 from CMC / Treh MNAs with loaded tips. 20x optical magnification. Composite fluorescence microscope images demonstrate the delivery wells penetrating the epidermis and upper dermis, and the delivery of antigen and adjuvant to the targeted cutaneous microenvironments.

[0035] FIG. 10A shows optical microscope images of MNAs integrating both Alexa555-OVA and Alexa488-Poly (I:C), with the fluorescent image being a representative figure of the pyramid head incorporating both charges.

[0036] FIG. 10B shows representative optical microscope images of Alexa555-OVA and Alexa488-Poly (I:C) loaded MNAs after in vivo application in the mouse shown.

[0037] FIGS. 10C and 10D show the effective co-administration of Alexa488-Poly (I: C) and Alexa555-OVA alongside skin microenvironments using new MNAs.

[0038] FIG. 11A illustrates representative histograms from flow cytometry analysis showing cells labeled with CFSE remaining in spleens of immunized and non-immunized mice.

[0039] FIG. 11B shows the quantification of specific cell lysis, with 100% lysis corresponding to the complete elimination of target cells (mean ± SD, 3 rats per group).

[0040] FIG. 11C shows the serum concentrations of OVA-specific IgG1 and IgG2c antibodies (the bars represent the mean values, 3 mice per group). DETAILED DESCRIPTION

[0041] The detailed descriptions in this document describe some illustrative embodiments related to the manufacture and Petition 870260056666, dated 11 / 06 / 2026, page 14 / 131 7 / 39 Use of microneedle arrays (MNAs). Although the illustrative embodiments may disclose particular types of MNAs, it should be understood that other types of MNAs may benefit from the systems and methods disclosed.

[0042] As used in this document, the term “biological”, “active component”, “bioburden” or “bioactive material” refers to pharmaceutically active agents, such as analgesic agents, anesthetic agents, anti-asthmatic agents, antibiotics, antidepressant agents, antidiabetic agents, antifungal agents, antihypertensive agents, anti-inflammatory agents, antineoplastic agents, anxiolytic agents, enzymatically active agents, nucleic acid constructs, immunostimulant agents, immunosuppressive agents, vaccines and the like. Bioactive material may comprise soluble materials, insoluble but dispersible materials, natural or formulated macro, micro and nano particulates and / or mixtures of two or more soluble, insoluble dispersible materials and natural and / or formulated macro, micro and nano particulates.In this regard, although a number of MNA examples disclosed here relate to vaccines and immunizations, any other suitable bioactive material, such as those discussed above, can be used in new MNA projects.

[0043] As used in this document, the term “pre-formed” means that a structure or element is manufactured, constructed and / or formed into a particular shape or configuration prior to use. Consequently, the shape or configuration of a pre-formed microneedle array is the shape or configuration of that microneedle array prior to the insertion of one or more of the microneedles of the microneedle array into the patient.

[0044] As used in this document, the term “relief” or “relief feature” refers to a sunken surface that is Petition 870260056666, dated 11 / 06 / 2026, p. 15 / 131 8 / 39 considered inaccessible using standard molding methods and, in particular, to a feature (e.g., a recess, protrusion or other geometric shape) that restricts or prevents the removal of a molded part with that feature from a conventional part mold.

[0045] The systems and methods described in this document, and their individual components, should not be interpreted as being limited to the particular uses or systems described in this document in any way. Instead, this disclosure is directed to all novel and non-obvious features and aspects of the various embodiments disclosed, alone and in various combinations and subcombinations with each other. For example, any features or aspects of the embodiments disclosed may be used in various combinations and subcombinations with each other, as will be recognized by one skilled in the art in the relevant field(s) in view of the information disclosed in this document.Additionally, the systems, methods, and their components disclosed are not limited to any specific aspect or feature or combination thereof, nor do the things and methods disclosed require that any one or more specific advantages be present, or problems be solved. The headings are provided for readability purposes only, and it should be understood that the elements and / or steps in one section may be combined with elements and / or steps under different headings in this disclosure.

[0046] As used in this application, the singular forms “a”, “an”, and “the” include the plural forms, unless the context clearly dictates otherwise. Additionally, the term “includes” means “comprises”. Furthermore, as used in this document, the term “and / or” means any item or combination of items in the sentence. Petition 870260056666, dated 11 / 06 / 2026, page 16 / 131 9 / 39 Additionally, the term “illustrative” means to serve as a non-limiting example, instance, or illustration. As used in this document, the terms “for example” and “by way of example” introduce a list of one or more non-limiting embodiments, examples, instances, and / or illustrations.

[0047] 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 form of description encompasses rearrangement unless a particular order is required by specific language set forth below. For example, operations described sequentially may, in some cases, be rearranged or performed simultaneously. Furthermore, for the sake of simplicity, the accompanying figures may not present the various ways in which the disclosed things and methods can be used in conjunction with other things and methods. Additionally, the description sometimes uses terms such as “provide,” “produce,” “determine,” and “select” to describe the disclosed methods. These terms are high-level descriptions of the actual operations that are performed.The actual operations corresponding to these terms will vary depending on the particular implementation and are readily discernible by one skilled in the art, having the benefit of this disclosure.

[0048] Illustrative Microneedle Arrangements

[0049] The use of microneedle arrays has several advantages over conventional needle-based injection techniques. For this reason, vaccination or administration of cutaneous medications using microneedle arrays offers a viable and attractive approach for effective immunization or immunotherapy against cancer due to the aforementioned theoretical advantages of the skin.

[0050] Unlike topical delivery approaches, the Petition 870260056666, dated 11 / 06 / 2026, p. 17 / 131 10 / 39 Microneedles (MNAs) physically penetrate the stratum corneum, thus eliminating formulation complexities and resulting in the localized deposition of vaccines or drugs in the skin's microenvironments. In contrast to traditional needle injections, microneedles are benign to pain receptors, allowing for minimally invasive, painless immunization.

[0051] Among other benefits, dissolving MNAs can provide highly effective vaccination due to their greater antigen loading capacity, adjustable release kinetics, simple fabrication, and long-term stability. Such MNAs can be created from water-soluble polymers that dissolve when inserted into the skin. The microneedles of the MNAs are preferably strong enough in their dry state to penetrate the stratum corneum and then rapidly dissolve in the fluid environment of the skin, thus releasing the vaccines. Precise administration of bioactive materials, such as vaccines, within cutaneous microenvironments can result in greater efficiency, thus requiring relatively smaller doses compared to traditional needle injections.

[0052] As disclosed in more detail below, microneedles with relief geometries that include dissolvable and / or non-dissolvable materials can be formed by the new methods and systems described in this document.

[0053] In some embodiments, the new MNAs can be formed via a single-step micromolding process that utilizes flexible production mold materials. The use of such flexible production mold materials allows the production of a variety of geometric designs that would otherwise not be possible. The flexible production mold material may comprise, for example, any elastomer or other material. Petition 870260056666, dated 11 / 06 / 2026, page 18 / 131 11 / 39 flexible that allows the removal of microneedles from a desired design (e.g., a desired amount of relief and / or other geometric shape). The ability to mold a variety of geometric shapes that would otherwise be difficult to manufacture by molding enables novel and innovative functionally graded MNAs, such as those shown in FIG. 1, for targeted delivery of a variety of bioactive materials to the skin and other tissues (e.g., cardiac and ocular tissue).

[0054] FIG. 1 illustrates examples of different microneedle structures that can be formed using the methods and systems described in this document. With reference to FIG. 1, four different microneedle structures 10, 12, 14, 16 are shown in a production mold 18 that is formed from a flexible material, such as a flexible elastomer.

[0055] The microneedle 10 is formed with a dissolvable support layer 20, a dissolvable shaft 22 and a microneedle tip 24 that is loaded with a bioactive material. Thus, the microneedle 10 is entirely manufactured from a dissolvable (or biodegradable) material. The bioactive material may be mixed with the dissolvable material, but preferably it is located at the tip of the needles, as shown in FIG. 1 to improve administration efficiency.

[0056] As shown in FIG. 1, microneedles 10, 12, 14, 16 may have a pyramid-shaped head with a sharp point and a recessed shaft that connects to a support layer via threaded bases. Threads 32 may provide enhanced mechanical performance during tissue insertion.

[0057] The MNAs described in this document may be manufactured from any moldable, biodegradable and / or biocompatible solvent materials that are not dissolvable, including Petition 870260056666, dated 11 / 06 / 2026, page 19 / 131 12 / 39 carboxymethylcellulose, trehalose, polyvinylpyrrolidone, poly(vinyl alcohol), maltodextrin, silk, glucose, hyaluronic acid, poly(methyl methacrylate), polycarbonate, poly(lactic-co-glycolic acid), poly(lactic acid), light-curable resins, and combinations thereof, to incorporate any bioactive materials, including cosmetics, dermal fillers, statins, growth factors, analgesics, antihistamines, vitamins, anesthetics, anti-aging agents, small molecule drugs, haptens, allergens, anti-inflammatory agents, proteins, peptides, microvesicles, exosomes, polyplexes (siRNA, shRNA, DNA vector complexes), recombinant viral vectors (i.e., adenovirus, lentivirus, vaccinia virus, adeno-associated virus and their different serotypes), monoclonal and polyclonal antibodies and live cells or Lisadas.

[0058] The microneedle 12 is formed with an insoluble support layer 26, an insoluble shaft 28 and a microneedle tip 24 that is loaded with a bioactive material. This functionally graduated relief MNA design can be manufactured with more than one material. For example, the pyramid part can be created using a solvent or biodegradable material, while the shaft part and the support layer are manufactured from a non-solvent material, such as a non-solvent biocompatible rigid polymer (e.g., poly(methyl methacrylate), polycarbonate, Vero White and other UV curable and heat curable resins).

[0059] The 12 microneedle can therefore provide a sharp needle tip along with enhanced mechanical performance through a filament base to allow successful tissue penetration and a pyramid head that serves as the dosing format for the bioactive material where the bioactive material(s) is incorporated into a dissolving or degradable biomaterial matrix. The embossed shaft enhances the Petition 870260056666, dated 11 / 06 / 2026, page 20 / 131 13 / 39 mechanical performance during penetration while ensuring tissue retention during implantation, and the insoluble relief stem portion prevents back-diffusion of the incorporated bioactive material(s) during both manufacturing and implantation processes. The insoluble support layer can also help prevent moisture absorption during storage, which can result in excessive bending of the support layers and render MNA applications less than ideal.

[0060] The 14 microneedle is similar in shape to the 12 microneedles, 14, but it also includes another dissolving layer 30. For example, the pyramid part is created using a biodegradable or solvent material and a faster dissolving layer is provided adjacent to the shaft / needle tip connection. The dissolving layer can be formed, for example, from a fast-dissolving polymer of low molecular weight, such as glucose, sucrose, trehalose, maltodextrin or polyvinylpyrrolidone. The rest of the shaft part and the support layer can be formed from an insoluble material, such as a rigid biocompatible insoluble polymer, such as acrylated polyesters, epoxies, UV curable monomers, resins, silicones.

[0061] The 14 microneedle, therefore, provides a sharp needle tip along with enhanced mechanical performance through a fluted base that allows successful tissue penetration, a pyramid head that serves as the dosing form for the bioactive material where the bioactive material(s) is incorporated into a dissolving or degradable biomaterial matrix, a raised shaft section enhances mechanical performance during penetration while ensuring tissue retention during implantation, and a fast-dissolving layer along with mechanical incompatibility between the Petition 870260056666, dated 11 / 06 / 2026, page 21 / 131 14 / 39 layers of dissolving and insoluble material, which facilitates the rapid separation of the pyramid tips.

[0062] The microneedle 16 is similar to the microneedle 14, but further comprises a corresponding support layer 34, which may be insoluble. In particular, the microneedle 16 may be formed with a pyramid portion that is created using a dissolvable or biodegradable material, the fast-dissolving layer may be formed as described above, the rest of the shaft portion may be manufactured from an insoluble material and the support layer may be manufactured from a conformable material, such as an insoluble conformable polymer (silicones, UV curable polymers, elastomers).

[0063] The microneedle 16 can therefore provide a sharp needle tip along with enhanced mechanical performance through a fluted base that allows successful tissue penetration, a pyramid head that serves as the bioactive material dosing form where the bioactive material(s) is incorporated into the degradation or dissolution biomaterial matrix, and a raised shaft portion that enhances mechanical performance during penetration while ensuring tissue retention during implantation. As with the microneedle 14, the fast-dissolving layer along with the mechanical mismatch between the dissolving and insoluble layers can facilitate rapid separation of the pyramid tips and the raised insoluble shaft portion and prevents back-diffusion of the incorporated bioactive material during both manufacturing and implantation processes.In this approach, the support layer can better conform to uneven skin topography and, if insoluble, can help prevent moisture absorption that can result in excessive curvature of the support layers and make them... Petition 870260056666, dated 11 / 06 / 2026, p. 22 / 131 15 / 39 MNA applications below ideal.

[0064] FIGS. 2A to 2C illustrate the ability of a microneedle arrangement 50 having microneedles 52 and an adaptable support layer 34 to bend and, after bending, return to an original state, or at least a state that is closer to the original state than the bent state.

[0065] It should be understood that any combination of the features disclosed in FIG. 1 - and throughout this application - is contemplated. For example, the adaptable support layer of microneedle 16 and the fast-dissolving layer 30 of microneedles 14, 16 can be used in combination with any of the other structures disclosed in this document (e.g., microneedles 10, 12).

[0066] Additionally, the processes disclosed in this document can be used to create a microneedle that is formed entirely of insoluble materials for use as coated MNAs, fabric adhesives (adhesives) and / or microprotrusions. Insoluble microprotrusion or microneedle geometries have been created previously using additive manufacturing or mechanical micromachining processes. However, high-throughput manufacturing of these geometries is hampered by the lack of effective micromolding processes.

[0067] Microadditive Manufacturing of MNAs

[0068] In some embodiments, MNAs can be formed using additive manufacturing (AM), including microadditive manufacturing (pAM) techniques. The methods and systems described in this document may allow medical researchers with little experience in microfabrication to directly produce their MNA designs from a computer-aided design (CAD) drawing without the complex requirements of manufacturing processes. Petition 870260056666, dated 11 / 06 / 2026, page 23 / 131 16 / 39 subtractive. The μAM techniques described in this document provide an effective means for the fabrication of novel MNAs specifically designed for the effective cutaneous and non-cutaneous delivery of drugs.

[0069] In some embodiments disclosed in this document, MNA designs included uniquely shaped micron-scale needles comprising sharp pyramid heads and recessed shafts with fluted bases to ensure successful skin penetration and retention. Unlike conventional MNAs, the MNAs disclosed in this document are manufactured by a three-dimensional (3D) μAM approach with 3D direct laser engraving, which offers transformative potential for the MNA field with its unparalleled level of simplicity and design capabilities.

[0070] As described in more detail below, in some embodiments, replicas of the main MNAs can be obtained from a strong mechanically moldable resin by a two-step, high-fidelity micromolding approach. These replicas can then be used to prepare production molds, such as polydimethylsiloxane (PDMS) production molds, which have enabled the fabrication of novel dissolvable MNAs loaded at the tip with raised microneedles. The resulting MNAs are, in some embodiments, fully dissolving MNAs with true raised features for effective cutaneous drug delivery.

[0071] As shown in FIG. 1, in some embodiments, a plurality of master MNAs can be printed, replicas of the master MNAs can be obtained quickly, and then a plurality of replicas can be assembled together to create larger MNAs.

[0072] In some forms, MNAs may incorporate a Petition 870260056666, dated 11 / 06 / 2026, page 24 / 131 17 / 39 Ovalbumin (OVA) model antigen ± Poly(I:C) model adjuvant from a biosoluble material composition (70% CMC / 30% Treh) using a spin casting method. Furthermore, the proposed MNA designs, along with the favorable mold materials, strategically allowed for direct fabrication steps without interfering with the molding processes. The fabricated MNAs are particularly effective at penetrating the stratum corneum of living human skin to deliver their bioburden to the cutaneous microenvironments. These unique MNAs meet the geometric and mechanical strength requirements for effective skin penetration for cutaneous vaccination, thus presenting a promising alternative approach for skin-targeted immunization strategies.

[0073] Microneedles and Illustrative Arrangements and their Systems of Manufacturing

[0074] FIGS. 3A and 3B illustrate an illustrative microneedle arrangement (MNA) 100 comprising microneedles with a pyramid head with a sharp tip 102 and a raised shaft portion 104. In addition, the microneedles include a threaded base 106.

[0075] The methods and systems disclosed in this document enable the reproducible fabrication of high-quality loaded tip dissolving MNAs with relief features from different and widely used dissolving microneedle materials, including CMC, PVP, Silk, HA, CMC / trehalose, CMC / Glucose, CMC / Sucrose, PVP / PVA and any other moldable biodissolving compositions.

[0076] In this modality, the microneedle has a height of 110 which is between 50 and 1500 pm, such as 750 pm in height, and a vertex angle A of the pyramid head which is between 10° -60, such as 30°. Petition 870260056666, dated 11 / 06 / 2026, p. 25 / 131 18 / 39

[0077] In one embodiment, the microneedle shaft portion may have a width 112 that is between 50 and 500 μm, such as 150 μm. The shaft portion 104 may extend from the lower region of a three-dimensional (3D) pyramid head to a microneedle support layer 108 with a threaded connection radius that varies from 15 to 75 μm, such as 35 μm. A width 114 of the lower region of the pyramid head may have, for example, between 100 and 400 μm, such as 250 μm x 250 μm base area.

[0078] In some embodiments, the tip-to-tip distance between the microneedles in the array can be between 100 and 800 μm, such as 650 μm. In some embodiments, the MNA can include between 1 and 1000 microneedles, such as 25 microneedles in a 5x5 array configuration on a support layer with an area of ​​4.75 mm x 4.75 mm.

[0079] The filament at the base of the 106 microneedle can help reduce mechanical stress concentrations associated with sharp corners and, in turn, enhances microneedle performance during manufacturing and skin insertion processes. The apex angle, width, and height of the microneedles were selected to provide improved skin insertion mechanics and to reduce penetration failures.

[0080] The relief, or anchor feature, can enhance skin retention during application and, using the new methods and systems disclosed in this document, can be achieved without interfering with processing steps, thus allowing direct removal of MNAs from the molds.

[0081] The three-dimensional microadditive manufacturing (3ϋ-μΑAM) approach described in this document allows the creation of unique MNA designs from a 3D-CAD drawing. Furthermore, 3DμΑAM can enable medical researchers with little or no... Petition 870260056666, dated 11 / 06 / 2026, page 26 / 131 19 / 39 no experience in microfabrication design and manufacture MNA-based drug delivery platforms with application-oriented optimizations.

[0082] Illustrative manufacturing systems and processes are presented in a flowchart in FIG. 4A and graphically illustrated in FIG. 4B. As shown in FIGS. 4A and 4B, the process can comprise the following steps to create new fast-dissolving MNAs while simultaneously achieving high-throughput manufacturing. First, a 3D-CAD design 132 of the MNA design can be prepared (process step 120). Second, a direct production of the master MNA 134 from the CAD design can be produced by direct 3D laser engraving using an insoluble resin (e.g., IP-S photoresist) (process step 122). The 3D-pAM manufacturing system can be, for example, a Nanoscribe 3D printing system 136.

[0083] Thirdly, a fast and high-fidelity replication of Master MNA can be formed with a UV-curable resin (e.g., VeroWhitePlus, Tangoblack, Digital Materials) using a two-step micromolding approach (process step 124). This approach may include a negative elastomer mold 138 to form the replica 140 of the master MNA.

[0084] Fourth, master MNA molds can be created with a plurality of master MNA replicas (e.g., six replicas) on 3D printed MNA supports (process step 126). The 3D printed MNA support 142 can be formed, for example, from a resin material. Combining a plurality of master MNA replicas together, for example, on 3D printed MNA supports, can allow the creation of larger MNAs and improve productivity.

[0085] Fifth, the production molds for MNA 144 can be Petition 870260056666, dated 11 / 06 / 2026, page 27 / 131 20 / 39 manufactured from PDMS elastomer using micromolding (process step 128). Sixth, dissolving MNAs with loaded tips 150 can be manufactured (process step 130). The MNAs can have embossed microneedles incorporating one or more bioactive materials, such as a vaccine or any other bioactive material from a water-soluble biocompatible material (e.g., a composition of CMC and Treh) through a multi-stage spin casting method using a centrifuge.

[0086] For example, a bioactive component (e.g., vaccine) can be spin-melted at the tip of PDMS production molds and the dissolvable hydrogel (e.g., CMC / Trehalose can be spin-fused into production molds to serve as the structural material of microneedles and to form the support layer of MNAs. In some embodiments, the master MNA, master MNA molds, including master MNA replicas, and elastomer production molds can be reused for a large number of processing cycles, thus significantly reducing manufacturing costs for MNAs with unique designs and enhancing productivity.

[0087] FIGS. 5A to 5E show final products corresponding to different manufacturing or processing stages. FIG. 5A shows an optical microscope image of the master MNA 134 created using 3D direct laser engraving, and FIG. 5D shows an optical microscope image of an individually embossed needle in the 3D printed master MNA 134. Specifically, the master MNA was manufactured from photoresist IP-S by 3D direct laser engraving. IP-S is a specific material designed for 3D laser lithography and provides high resolution and mechanical integrity for micro and nanostructures. Petition 870260056666, dated 11 / 06 / 2026, page 28 / 131 21 / 39 Two-photon polymerization-based 3D laser lithography provided an effective means for fabricating MNA designs with relief, smooth edges and sharp points, and without any unwanted residue (e.g., machining chips).

[0088] FIG. 5B shows an optical microscope image of replica 140 of the master MNA created using a two-stage micromolding strategy: elastomer molding combined with UV-curable micromolding, and FIG. 5E shows an optical microscope image of an individual embossed needle in replica 140 of the master MNA. The master MNA was produced using a two-stage micromolding process. The IP-S master MNA was used to fabricate a flexible PDMS mold via elastomer molding, and then the PDMS mold was used to fabricate multiple VeroWhite MNA replicas via UV-curable micromolding. VeroWhite resin is a wear-resistant acrylic-based photopolymer that can be used with Polyjet 3D printers.

[0089] To achieve scalable manufacturing of new MNAs, MNA replicas were then used to create MNA master molds that include multiple MNAs (e.g., six MNA replicas). The final MNA master molds were post-processed in a vacuum oven to facilitate successful curing of PDMS on the mold surface, and then elastomer MNA production molds, which consisted of microneedle-shaped wells, were fabricated from PDMS. FIG. 5C shows an optical microscope image of the microneedle-shaped wells in MNA production molds 144.

[0090] Thus, these processing steps, together with the high geometric capability of direct 3D laser engraving, resulted in a scalable and effective MNA manufacturing strategy. Furthermore Petition 870260056666, dated 11 / 06 / 2026, page 29 / 131 22 / 39 In addition, rapid replication of the 3D-printed master MNA using a wear-resistant moldable material has substantially improved productivity. Other relief features using different microneedle and elastomer mold materials, consistent with the processes and systems described in this document, are also possible.

[0091] When manufacturing the master MNA molds with six MNA replicas, the dissolving MNAs that integrate the vaccine in the needle tip portion were manufactured using the conventional three-stage manufacturing strategy through the master mold to the production mold for the final dissolving MNAs.

[0092] As discussed in more detail below, dissolving MNAs incorporating the vaccine (10 pg OVA + 25 pg Poly (I: C)) in the tip portion of the embossed needles were fabricated via spin casting from two different material compositions (i.e., CMC / Trehalose and PVP / PVA). Furthermore, CMC / Trehalose MNAs with embossed microneedle-loaded tips integrating a colored model drug (e.g., Doxorubicin) were fabricated to facilitate image representation and demonstrate compatibility with chemotherapeutic agents. The systems and methods described in this document enabled the rapid and efficient fabrication of dissolving MNAs with embossed microneedle-loaded tips from different dissolvable material compositions.

[0093] FIG. 6A shows an optical microscope image of Final dissolving MNAs (CMC / Treh) incorporating bioloaders (e.g., OVA + Poly(I:C)). FIG. 4B shows an optical microscope image of PVP / PVA dissolving MNAs with a tip loaded with embossed microneedles integrating OVA, and FIG. 4C shows an optical microscope image of a microneedle. Petition 870260056666, dated 11 / 06 / 2026, p. 30 / 131 23 / 39 embossed CMC / Treh tip with loaded tip incorporating Doxorubicin as a colored drug to facilitate image representation.

[0094] Example 1 - Manufacturing of Illustrative MNAs

[0095] An illustrative process for creating a NAM is described in this document.

[0096] Master MNA Fabrication. A unique MNA project was created directly from the 3D-CAD design shown in FIG. 1 using 3D laser printing (Nanoscribe Photonic Professional, GT) with IP-S photopolymer resistance. The Nanoscribe printing system was equipped with a laser generator, an optical cabinet, a Zeiss optical microscope coupled with a lens to focus the laser beam, a Galvo mirror system to direct the laser beam scan, a piezoelectric stage for precise motion control, and operating software (Nanowrite) to execute the 3D printing. The entire system was placed on an optical table to eliminate vibrations during the printing process.

[0097] To manufacture the master MNA, the MNA design was converted into an 'STL' (Stereolithography) format. Subsequently, the STL file was loaded into the specialized software (DeScribe, Germany) of the Nanoscribe system to select the processing conditions (i.e., slicing distance, incubation, and splitting). Finally, the STL file was converted to the 'GWL' (General Writing Lithography) format to be exported in the Nanowrite software to print the master MNA. The master MNA was fabricated using Galvo-scan mode in the XY plane and piezo-scan mode in the Z direction. The master MNA was divided into 220 pm x 220 pm x 200 pm blocks within the working range and then stitched together. The laser power and engraving speed were set at 100 mW and 6 cm / s, respectively. The distances of Petition 870260056666, dated 11 / 06 / 2026, page 31 / 131 24 / 39 minimum and maximum cuts of 0.3 µm and 0.5 µm, respectively, were used. The master MNA was then printed by two-photon polymerization of the IP-S photoresist by a pulsed femtosecond laser at a wavelength of 750 nm using a unique deep liquid mode with the 25x objective and NA0.8 in Shell and Scaffold mode.

[0098] After printing, the MNA master was developed in the photoresistant solvent propylene glycol ether methyl acetate (PGMEA) for 30 min, followed by a 5 min rinse with isopropyl alcohol (IPA). After the MNA master was air-dried, it was placed under a UV light (365 nm) with an intensity of 16 mW / cm2 for 30 min to further crosslink the body to strengthen the MNA structure.

[0099] Master MNA Replication. To replicate the master MNA with high fidelity using a UV-curable resin, a two-stage micromolding method was performed. First, an elastomer mold, which is negative with respect to the master MNA, was fabricated from polydimethylsiloxane (PDMS) using a micromolding approach. Elastomer molding using PDMS provides accurate and reproducible replication of high-fidelity micron-scale structures. Briefly, the master MNA was mounted on a 5 cm diameter petri dish, and the PDMS was obtained using the two-component curable silicone elastomer, SYLGARD® 184 (Dow Corning), by mixing the base material with a curing agent in a SYLGARD® to curing agent ratio of 10:1. Subsequently, the mixture was poured over the mounted master MNA in the petri dish and degassed for 15 min.Next, the MNA masterbatch 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 minutes and then separated from the MNA masterbatch to obtain the negative PDMS mold. Petition 870260056666, dated 11 / 06 / 2026, page 32 / 131 25 / 39

[00100] The second processing step utilized the negative PDMS mold for the fabrication of positive MNA replicas from UV-curable resin (Stratasys®, VeroWhiteplus-RGD835). For each PDMS mold, 20 pl of liquid (uncured) resin was poured onto the molds, and then the molds were placed in a centrifuge to fill the microneedle-shaped wells with the resin at 4500 RPM and 20°C for 1 min. The resin was then cured under UV light (365 nm) at an intensity of 21.7 mW / cm² for 5 min on each of the top and bottom sides to cure both the base and the tips of the microneedle. To ensure that the support layers of the master MNA replicas were flat, a secondary loading of 50 pl of UV-curable resin was performed on the PDMS mold, which was the excess of the remaining available volume. A sheet of glass was placed over the mold to remove excess VeroWhite resin, thus creating a uniform, flat surface on the base.The liquid resin was then cured from the top side for 5 minutes and then demolded to obtain replicas of the master MNA.

[00101] Creation of MNA Master Molds. To facilitate the scalable manufacturing of dissolving MNAs, MNA master molds were created by assembling six replicas of the MNA master onto MNA supports manufactured by Stratasys® from an insoluble photopolymer (Vero White) using a high-resolution (16 µm) Polyjet 3D printing system (Objet Connex 500 multimaterial). The 3D model of the MNA supports was created using SolidWorks 2018 CAD software and then converted to the 'STL' (Stereolithography) file format. Subsequently, specialized software (Objet Studio) divided this 3D model into 2D cross-sectional layers, creating a computer file that was sent to the 3D printer system. The channels in the MNA support Petition 870260056666, dated 11 / 06 / 2026, page 33 / 131 26 / 39 3D printed molds were designed and manufactured to serve as raised pockets in MNA production molds to assist as reservoirs for both the bioactive payload (e.g., vaccine) and the structural hydrogel material of dissolving MNAs during the spin casting process. The created master MNA molds were baked at 80°C overnight in a vacuum oven to facilitate the efficient fabrication of elastomer MNA production molds.

[00102] Fabrication of MNA production molds. MNA production molds that included microneedle-shaped wells were fabricated from a commonly used elastomer polydimethylsiloxane (PDMS) as described for master MNA replication. The base material was mixed with a curing agent in a ratio of SYLGARD® to curing agent of 10:1. Subsequently, the mixture was poured onto the master MNA mold placed in a 10 cm diameter petri dish and degassed for 15 min. Then, the master mold with the degassed mixture was placed in an oven to cure PDMS at 70 °C for 1 h. After cooling the cured PDMS to room temperature, it was separated from the master MNA mold to fabricate the PDMS MNA production molds.

[00103] Production of dissolving MNAs. For dissolving MNAs with a microneedle-loaded tip incorporating OVA ± Poly (I : C), a biosoluble material composition that included carboxymethylcellulose (CMC, cat # C5678, Sigma-Aldrich, St. Louis, MO) and trehalose (Treh, Cat # T9531, Sigma-Aldrich) were used. To prepare the hydrogel form of the dissolving MNA structural material, CMC and Treh powders were thoroughly mixed based on a weight ratio of 70% and 30%, respectively. This powder mixture was then added Petition 870260056666, dated 11 / 06 / 2026, page 34 / 131 27 / 39 endotoxin-free water (HyClone HyPure Cell Culture Grade Water) was thoroughly mixed to achieve a solute concentration of 30% w / w. The prepared hydrogel was refrigerated at 4°C for 24h to allow the mixture to equilibrate. CMC / Treh MNAS with uniquely designed loaded tips were then fabricated using a multi-stage spin casting technique with a centrifuge (Thermo Fisher Scientific Sorvall Legend XTR with TX-750 oscillating bucket rotor).

[00104] First, 5 pL of OVA solution (25 mg / mL of OVA in endotoxin-free water) were dispensed onto each of the MNAs in the PDMS production molds, and the production molds were centrifuged for 1 min at 20 °C and 4500 rpm to fill the microneedle-shaped cavities. Once the production molds were filled, the excess OVA solution within the reservoir was recovered. The production molds were again centrifuged for 30 min at 20 °C and 4500 rpm to ensure that the dry OVA load was localized to the tip portion of the needle-shaped cavities in the production molds.

[00105] For MNAs integrating OVA + Poly(I:C), after OVA loading, 5 pL of Poly(I:C) solution (62.5 mg / mL Poly(I:C) in endotoxin-free water) was dispensed onto each MNA in the PDMS production molds, and the production molds were centrifuged for 1 min at 20°C and 4500 rpm to fill the microneedle-shaped cavities. Once the production molds were filled, the excess Poly(I:C) solution within the reservoir was recovered. The production molds were again centrifuged for 30 min at 20°C and 4500 rpm to ensure that the Poly(I:C) load was also located in the pyramid portion of the needle-shaped cavities in the production molds. After locating the bioloads at the tip of the Petition 870260056666, dated 11 / 06 / 2026, page 35 / 131 28 / 39 microneedles, 40 pL of hydrogel (30% w / w 70:30 CMC:Treh) were loaded onto each of the MNAs in the PDMS production molds to fill the microneedle-shaped geometries in the production molds and to form the support layers for the MNAs. The hydrogel-loaded production molds were then centrifuged for 5 h at 20 °C and 4500 rpm to obtain dissolving MNAs loaded with 10 pg OVA + 25 pg Poly(I:C). Due to the specific geometry of the MNA production molds, each replication produced six arrays of dissolving MNAs loaded with OVA ± Poly(I:C) simultaneously.

[00106] To demonstrate the fabrication of dissolving MNAs with embossed microneedles from another water-soluble material (i.e., material capacity), a biosoluble polymer composition of Polyvinylpyrrolidone (PVP) and polyvinyl alcohol (PVA) (40% w / w 60:40 PVP:PVA) was also used to fabricate MNAs with different bioloads. The fabricated master MNAs, MNA replicas, elastomer MNA production molds, and OVA ± Poly(I:C) loaded dissolving MNAs were photographed using bright-field optical microscopy to assess the geometric integrity of the novel embossed microneedles.

[00107] Cutaneous Administration Using Illustrative MNAs

[00108] Preparation of ex vivo human skin explants. Human skin explants were prepared from unidentified healthy donors undergoing plastic surgery, acquired through the Pitt Biospecimen Core, and used according to University of Pittsburgh Medical Center guidelines. The tissue was rinsed in 70% ethanol and then in phosphate-buffered saline (PBS). Human skin explants (approximately 1 mm thick) were harvested using a... Petition 870260056666, dated 11 / 06 / 2026, page 36 / 131 29 / 39 miniature skin graft knife from Silver (Padgett, Integra Miltex, Plainsboro, NJ) and then cut into 20 mm x 20 mm square pieces. The resulting human skin samples consisted of unaltered epidermis and a thin layer of underlying dermis, and were maintained as explants in a normal physiological state by culture at an air-fluid interface.

[00109] Image analysis. To evaluate the administration of intradermal bioactive materials directed by MNAs (e.g., vaccine) in live human skin explants, a series of image analyses were performed. CMC / Treh MNAs with a loaded tip incorporating a colored load (i.e., Allura Red R40 dye) were fabricated using the fabrication strategy described above. Before applying the MNAs to the human skin explants, the MNAs were photographed using bright-field optical microscopy.

[00110] Subsequently, MNAs were applied to human skin explants and removed after 10 min. The targeted regions of human skin were then examined under a bright-field microscope to image the patterns of colored bioburden deposited from the CMC / Treh MNAs on human skin. The remaining MNA materials were also photographed using optical microscopy after application to human skin. For further qualitative evaluation of MNA-directed intradermal vaccine administration to human skin, CMC / Treh MNAs incorporating Alexa555-labeled OVA and Alexa488-labeled Poly(I:C) were fabricated, applied to human skin for 10 min, and removed. The targeted human skin explants were then analyzed histologically. Briefly, MNA-treated human skin samples were fixed in 2% paraformaldehyde followed by immersion in sucrose solution, with 3 changes of this solution in 24h.The tissue sections were then rapidly frozen. Petition 870260056666, dated 11 / 06 / 2026, page 37 / 131 30 / 39 composite of ideal sectioning temperature (OCT) histology and cryosectioned into sections approximately 10 μm thick. The sectioned human skin samples were counterstained with nuclear fluorescent dye DAPI. The stained sections were then photographed using a Nikon transmission fluorescence microscope to detect Alexa555-OVA and Alexa488-Poly (I:C) in cross-sections of human skin, as well as using a bright-field microscope to better demonstrate stratum corneum violation.

[00111] MNA-directed cutaneous immunization in vivo.

[00112] Mice and animal husbandry. Female C57BL / 6J mice were acquired from The Jackson Laboratory (Bar Harbor, ME) and used at 8–10 weeks of age. Mice were kept under specific pathogen-free conditions at the University of Pittsburgh, and all experiments were conducted in accordance with the Institutional Animal Care and Use Committee (IACUC) guidelines.

[00113] IVIS in vivo image. In vivo intradermal vaccine administration using novel dissolving MNAs was demonstrated using a C57BL / 6J mouse. MNAs of CMC / Treh loaded tips integrating both Alexa555-labeled OVA and Alexa488-labeled Poly(FC) were created using the fabrication strategy described above and applied to the abdomen of a mouse that was temporarily anesthetized by isoflurane inhalation for 10 min. The MNAs were then removed, and the mouse returned to normal activity. OVA + Poly(I:C) loaded MNAs were photographed before and after in vivo application. The image of the MNA-treated mouse was loaded into the IVIS 200 in vivo imaging system (PerkinElmer) using filters to test for fluorescently labeled Poly(I:C) and OVA at the MNA application site. The image was post-Petition 870260056666, dated 11 / 06 / 2026, p. 38 / 131 31 / 39 processed using Living Image software (PerkinElmer).

[00114] Cell-mediated and humoral immune responses. To demonstrate cutaneous vaccination with novel MNAs, CMC / Treh MNAs with a loaded tip integrating 10 pg OVA + 25 pg Poly(I:C) were prepared as described above. Mice were immunized by cutaneous vaccination using MNAs (10 pg OVA + 25 pg Poly(I:C), MNAs applied to the right and left sides of the abdomen, two MNAs per mouse) or by two intramuscular injections of 10 pg OVA in PBS into the gastrocnemius muscle of the hind limb or left untreated (i.e., naive). Immunizations were performed on days 0 and 7 as initial and booster doses, respectively. Mice were then tested for OVA-specific cytotoxic T cell activity in vivo and OVA-specific antibody response 5 days after the booster dose using well-established techniques.

[00115] For the OVA-specific antibody response, blood was collected from anesthetized mice at the time of sacrifice by cardiac puncture, and serum was isolated using BD Microtainer serum separator tubes (BD Biosciences, San Jose, CA). OVA-specific IgG1 and IgG2c antibodies in serum were measured by indirect ELISAs. Costar EIA / RIA plates (Corning Inc., Corning, NY) were coated with OVA (100 pg / mL in 0.5 M carbonate-bicarbonate buffer, pH 9.6; Sigma) by overnight incubation at 4 °C. The plates were washed (3x) with 0.05% Tween20 in PBS and blocked with 1% goat serum in PBS for 1 hour at 37 °C. Serum samples and standards (IgGl anti-OVA from Cayman Chemical, Ann Arbor, MI; IgG2c anti-OVA from Chondrex, Redmond, WA) were diluted with 1% goat serum, added to plates, and incubated for 2 hours at 37°C. After washing (3x), the plates were incubated for 1 hour at 37°C with secondary antibodies. Petition 870260056666, dated 11 / 06 / 2026, p. 39 / 131 32 / 39 biotinylated (anti-mouse goat IgG or IgG2c, 1:20,000 in 1% goat serum; Jackson ImmunoResearch, West Grove, PA). The plates were then washed (3x) and incubated for 30 min with streptavidin-HRP (1:1000 in 1% goat serum; BD Biosciences). The plates were washed (3x) again and incubated at room temperature with 4,4',5,5'-tetramethylbenzidine (TMB) peroxidase substrate (Sigma) for 2 to 3 minutes and the reaction was quenched with 1.0 M H2SO4. For all ELISAs, absorbance at 450 nm (OD450) was read with a SpectraMax 340PC plate reader (Molecular Devices, Sunnyvale, CA), and serum concentrations were calculated from standard curves.

[00116] To evaluate the activity of OVA-specific cytotoxic relief cells (CTLs), splenocytes from young mice were pulsed with 2 pg / ml of OVA257-264 peptide (SIINFEKL), or left unpulsed for 1 h. Splenocytes pulsed with antigen were washed and stained with high-concentration carboxyfluorescein succinimidyl ester (CFSE, 10 μM), while unpulsed splenocytes were labeled with low-concentration CFSE (1 mM) for 15 min at 37 °C. A 1:1 mixture of pulsed target cells and unpulsed control cells (107 each) was injected intravenously (IV) into immunized and young mice. Twenty hours after adoptive transfer, the spleens of the mice were isolated and target cell death was assessed by comparing pulsed and non-pulsed antigen populations using flow cytometry to quantify OVA-specific death of targets pulsed with high-CFSE-labeled SIINFEKL.Specific lysis was calculated and expressed as a percentage of maximum lysis as % lysis = {1 - [(average CFSElow / CFSEhigh ratio of young mice) / (CFSElow / CFSEhigh ratio of vaccinated mice)]} x 100. Petition 870260056666, dated 11 / 06 / 2026, p. 40 / 131 33 / 39

[00117] Intradermal and non-cutaneous uses

[00118] In addition to cutaneous vaccination, the MNAs described in this document can be used for a wide variety of intradermal and non-cutaneous drug delivery applications (e.g., ocular and cardiac tissues). As shown in FIGS. 7A to 7D, the bioburden of interest can be located at the tip of the microneedles in the spin casting process (i.e., a part of a pyramid) or the entire pyramid region can be loaded with the bioburden depending on the dose requirements. FIG. 7A shows PVP / PVA MNAs incorporating Dextran labeled with Texas Red (molecular weight of approximately 40 kDa) at the microneedle tips, while FIG. 7B shows CMC / Treh MNAs with loaded tips integrating Allura Red R40 dye (molecular weight of approximately 500 Da) in the pyramid region of the microneedles.

[00119] In addition, a series of repeated spin casting steps can be performed to manufacture high-quality embossed microneedle MNAs incorporating various fillers in their pyramid regions (FIGS. 7C and 7D). FIG. 7C shows loaded-tip PVP / PVA MNAs incorporating various fillers such as Texas Red labeled Dextran and Allura Red R40 dye, and FIG. 7D shows loaded-tip PVP / PVA MNAs incorporating various fillers such as Texas Red labeled Dextran and Alexa488 labeled PLGA microparticles (average diameter of 10 µm).

[00120] Thus, the approach presented and the new MNA designs are compatible with single and combined therapies for various cutaneous and non-cutaneous applications. It is important to emphasize that the same production molds can be used to effectively manufacture microneedle dissolution MNAs with Petition 870260056666, dated 11 / 06 / 2026, p. 41 / 131 34 / 39 relief for multiple cycles (e.g., FIGS. 7A and 7C show the MNAs obtained after the first and twelfth cycles using t, respectively). More importantly, in vivo cutaneous vaccination applications of single-load (OVA) and multiple-load (OVA + Poly (I : C)) MNAs were demonstrated in the current study.

[00121] Additive manufacturing or 3D printing, as used in the systems and methods described in this document, provides precise and reproducible fabrication of complex 3D geometries without design limitations and offers a high degree of design flexibility and control. Using the systems and methods in this document, a rapid turnaround from design to manufacturing for ideal application-oriented drug delivery systems is possible.

[00122] Various Needle Geometries

[00123] As discussed above, the systems and methods disclosed in this document offer an unprecedented level of design flexibility for MNA projects. To demonstrate the variety of geometric capabilities of direct 3D laser engraving, microneedle designs were fabricated with diverse geometries.

[00124] Figures 8A to 8B show that this technology allows the fabrication of a wide variety of microneedle geometries with high fidelity for application-oriented optimization. Furthermore, as shown in Figures 8A to 8B, it allows for a wide range of design changes, including height, width, vertex angle, and microneedle geometry without the need for complex and customized processing steps. Thus, this technology paves the way for the design and fabrication of unique, application-focused MNA designs.

[00125] Administration of intradermal vaccine based on MNA for human skin

[00126] To evaluate the cutaneous administration characteristics of Petition 870260056666, dated 11 / 06 / 2026, page 42 / 131 35 / 39 bioloads of novel MNAs with embossed needles, MNAs with Allura Red R40 dye-loaded tips, were fabricated using the fabrication strategy presented. Human skin explants were prepared as described above. The MNAs loaded with Allura Red R40 dye were applied to live human skin explants and removed after 10 min. Images of these MNAs before (FIG. 9A) and after (FIG. 9B) application demonstrated high-quality MNAs and complete dissolution of the microneedles, respectively. The corresponding deposits of embedded MNA load (e.g., Allura Red R40 dye) in the target skin are shown in FIG. 9C.

[00127] Successful vaccine administration through the stratum corneum in skin microenvironments rich in immune cells is critical for effective intradermal immunization. To anatomically evaluate the administration of OVA and Poly(I:C) in human skin, CMC / Treh MNAs incorporating both Alexa555-labeled OVA and Alexa488-labeled Poly(I:C) were applied to human skin explants for 10 min and then removed. The target human skin was cryosectioned and photographed using a Nikon transmission fluorescence microscope. Histology demonstrated microneedle pits penetrating through the epidermis into the dermis (FIG. 9G), and the administration of fluorescently labeled OVA and Poly(I:C) within the target human skin microenvironments (as FIG.9D to 9I: DAPI nuclear staining, Poly (I:C) labeled Alexa488, OVA labeled Alexa555, Brightfield, overlay of 3 different fluorescent colors, merged images of all fluorescent images with the brightfield image, respectively). In FIGS. 9D to 9I, the scale bars correspond to 100 pm.

[00128] Together, these images suggest that the unique MNAs presented met the geometric requirements (i.e., points). Petition 870260056666, dated 11 / 06 / 2026, page 43 / 131 36 / 39 sharp and smooth edges) and mechanical strength for flawless penetration into human skin (i.e., disruption of the stratum corneum and viable epidermis) and material requirements for efficient dissolution in the aqueous medium of the skin, thus providing an effective cutaneous platform for drug and vaccine delivery.

[00129] MNA-directed skin immunization in vivo

[00130] To study intradermal vaccine administration using novel MNAs in vivo in mice, CMC / Treh MNAs with high-fidelity embossed microneedles incorporating Alexa555-labeled OVA and Alexa488-labeled Poly(I:C) were fabricated as described above. Prior to application, Alexa555-OVA + Alexa488-Poly(I:C) MNAs were photographed using bright-field optical microscopy and epi-fluorescence microscopy (FIG. 10A). The MNAs were then applied to mice and removed after 10 min. The remaining MNA material after application was also photographed using bright-field optical microscopy (FIG. 10B). MNA-treated mice were photographed using the IVIS 200 live animal imaging system with filters for Alexa488-Poly(I:C) and Alexa555-OVA. The co-administration led by MNA of Poly (I : C) and OVA was presented in FIGS. 10C and 10D, respectively.Together, these images demonstrated the successful application of novel MNAs in vivo in mice and, in turn, the effective cutaneous drug and vaccine delivery conducted by MNAs with novel MNA designs.

[00131] After demonstrating successful intradermal administration of the vaccines in vivo, we specifically evaluated the immunogenicity of the antigen embedded in MNA ± adjuvant and compared immunization with MNA to vaccination via the clinically common intramuscular (IM) injection route. For this purpose, CMC / Treh MNAs were manufactured with 10 pg of OVA + 25 pg of Poly (I:C) Petition 870260056666, dated 11 / 06 / 2026, page 44 / 131 37 / 39 by MNA as described above. We and others have previously shown that dissolving integrated MNA proteins maintained their integrity. For immunization, the vaccination regimen described in the methods section was followed for immunization of IM and MNA mice via their abdomens. Cytotoxic T cell (CTL) and OVA-specific antibody responses were quantified using the standard in vivo lytic assay and ELISA, respectively. Cutaneous vaccination with MNAs triggered robust antigen-specific cellular immune responses (FIGS. 11A to 11B). As expected, equivalent numbers of antigen-pulsed target cells (CFSEhigh) and non-pulsed target cells (CFSElow) were recovered from naive or non-immunized mice (FIG. 11A), thus indicating the absence of antigen-specific cytolytic activity.In contrast, antigen-pulsed target cell-specific lysis was dramatically increased in immunized mice, as shown by the reduced survival of OVA-pulsed targets compared to non-pulsed targets. Specifically, while minimal differences in recovery between the two populations were observed in IM-OVA immunized mice (FIG. 11A), OVAMNA-immunized mice demonstrated greater OVA-specific lysis in vivo, with significantly (compared to IM-OVA) lower survival and recovery of OVA-pulsed targets than non-pulsed targets (FIG. 11A). Immunization with OVA + Poly(I:C) further enhanced vaccination performance (FIG. 11A). Quantification of antigen-specific lysis by standard techniques confirmed that MNA immunization induced potent CTL immunity (FIG. 11B), which was further enhanced by the inclusion of the Poly(I:C) adjuvant.In comparison, IM immunization induced low-level responses compared to non-immunized controls. Taken together, these... Petition 870260056666, dated 11 / 06 / 2026, page 45 / 131 Results 38 / 39 demonstrate that these MNAs can efficiently deliver antigens + adjuvants to APC-rich microenvironments within the skin to induce potent CTL immune responses.

[00132] In addition to CTL immunity, cutaneous vaccination with MNAs triggered robust antigen-specific humoral immune responses (FIG. 11C). While vaccination of mice with MNAs and IM resulted in IgG responses, those vaccinated with dissolving MNAs had significantly higher IgG titers, thus indicating the importance of MNA technology in immunization efforts. Ultimately, the novel presented MNAs provide a unique opportunity for the specific and precise delivery of embedded antigen ± adjuvant to defined microenvironments within the skin. For example, we and others have demonstrated that the skin is rich in dendritic cells and other antigen-presenting cells (APCs) essential for vaccine-induced immune induction.Therefore, targeting skin APCs using MNAs can be an effective strategy for vaccination in general and, in particular, for inducing cell-mediated immune responses, including CTL responses essential for preventing or treating many infectious diseases and cancer. Thus, the presented MNA technology may enable relevant capabilities for a wide variety of vaccination strategies.

[00133] Consequently, the development and application of novel dissolving microneedle MNAs with raised needles provides effective intradermal vaccination. The unique MNA designs include pyramid heads and raised rod regions with fluted bases. The manufacturing approach to create the raised MNAs strategically involved 3D laser printing and a series of micromolding processes. The successful and reproducible fabrication of dissolving microneedle MNAs with raised needles that Petition 870260056666, dated 11 / 06 / 2026, page 46 / 131 The 39 / 39 model incorporating a myriad of bioburdens was achieved using different biocompatible and water-soluble polymers. More importantly, the unprecedented level of geometric capability with 3D laser printing was demonstrated in application-driven MNA designs for various cutaneous and non-cutaneous drug delivery applications. The novel MNAs introduced with relief features met the resistance requirements for flawless skin penetration in humans and mice, and successfully delivered their bioburdens to targeted cutaneous microenvironments. Importantly, cutaneous vaccination using the antigen-loaded MNA model (OVA-MNA) elicited significantly more potent antigen-specific cellular and humoral immune responses than those obtained by traditional intramuscular injection-based immunization (IM-OVA).Most importantly, MNA-directed co-administration of the antigen (OVA) with the adjuvant (Poly(I:C)) enhanced the immunogenicity of the vaccine compared to IM-based vaccination with respect to enhanced MNA-directed cutaneous vaccination. Together, the approach presented provides an effective means of manufacturing novel dissolving MNAs for a wide variety of cutaneous and non-cutaneous drug delivery applications.

[00134] In view of the many possible embodiments in which the principles of the disclosed invention may be applied, it should be recognized that the embodiments illustrated are only preferred examples of the invention and should not be taken as limiting the scope of the invention. Instead, the scope of the invention is defined by the following claims. Therefore, we claim as our invention everything that is within the scope and spirit of these claims. Petition 870260056666, dated 11 / 06 / 2026, p. 47 / 131

Claims

1 / 8 CLAIMS 1. Method for forming an arrangement of microneedles (50, 100, 150), characterized in that it comprises: forming a production mold (18, 144) from a flexible material, the production mold (18, 144) comprising a plurality of cavities, with each of the pluralities of cavities being molded to define a plurality of respective microneedles (10, 12, 14, 16, 52), each having a shaft (22, 28, 104), a microneedle tip (24, 102), a threaded base (32, 106), and at least one relief feature (104), delivering a biodegradable matrix into each of the pluralities of cavities of the production mold (18, 144) to form the shaft (22, 28, 104), the microneedle tip (24, 102) and the threaded base (32, 106) of each respective microneedle, wherein at least one bioactive material is incorporated into at least one portion of the biodegradable matrix, the biodegradable matrix comprising a first dissolvable material;form a support layer (20, 26, 34, 108) adjacent to a lower portion of the rod (22, 28, 104); and remove the microneedles (10, 12, 14, 16, 52) from the production mold (18, 144) by pulling the microneedles (10, 12, 14, 16, 52) out of the mold, wherein at least one portion of the biodegradable matrix including at least one bioactive material is delivered into at least one microneedle tip (24, 102) defined by the respective production mold cavities (18, 144), wherein the flexible material has sufficient elasticity to allow the molded microneedle arrangement (50, 100, 150) to be removed from the production mold (18, 144) in a single pull without damaging the integrity of the shape of the microneedles (10, 12, 14, 16, Petition 870260056666, dated 11 / 06 / 2026, p. 64 / 131 2 / 8 52) as defined by the mold, and wherein the support layer (34, 108) is formed from a conformable material.; 2. Method according to claim 1, characterized in that at least one relief feature (104) is directly below the tip of the microneedle (24, 102).

3. Method according to claim 1 or 2, characterized in that the rod (22, 28, 104) is formed from the first soluble material or from an insoluble material.

4. Method, according to any one of claims 1 to 3, characterized in that the formation of the plurality of microneedles (10, 12, 14, 16, 52) comprises providing a second dissolvable material in the production mold (18, 144) to form a dissolution layer (30) on a part of the rod (22, 28, 104) that is adjacent to the tip of the microneedle (24, 102), optionally wherein the second dissolvable material of the dissolution layer (30) is selected from a material that dissolves more rapidly than the biodegradable matrix containing the bioactive material.

5. A method according to any one of claims 1 to 4, characterized in that the first dissolvable material comprises carboxymethylcellulose, trehalose, polyvinylpyrrolidone, maltodextrin, silk, hyaluronic acid, poly(vinyl alcohol), polyethylene glycol, poly(lactic-co-glycolic acid), poly(lactic acid), or a combination thereof.

6. Method according to claim 4, characterized in that the second dissolvable material comprises a fast-dissolving polymer of small molecular weight, optionally wherein the second dissolvable material is glucose, trehalose, sucrose, maltodextrin, polyvinylpyrrolidone or a combination thereof.

7. Method according to claim 1, characterized in that the conformable material of the support layer (34) is an insoluble polymer that can flex, deform or bend to conform to the non-uniform topography of the skin.

8. A method, according to any one of claims 1 to 7, characterized in that at least one bioactive material comprises two or more bioactive materials.

9. A method, according to any one of claims 1 to 8, characterized in that one or more bioactive materials comprise cosmetics, dermal fillers, statins, growth factors, analgesics, antihistamines, vitamins, anesthetics, anti-aging agents, small molecule drugs, haptens, allergens, anti-inflammatory agents, proteins, peptides, microvesicles, exosomes, polyplexes (siRNA, shRNA, DNA vector complexes), recombinant viral vectors (i.e., Adenovirus, Lentivirus, Vaccinia Virus, Adeno-associated Virus and their different serotypes), monoclonal and polyclonal antibodies and live or lysed cells.

10. Method, according to any one of claims 1 to 9, characterized in that the plurality of microneedles (10, 12, 14, 16, 52) has a shape that includes a circular obelisk, a square obelisk, a beveled obelisk, a pyramid on a square rod, an arrowhead shape on a rod, a pyramid head on a square obelisk rod, a conical head on a circular obelisk rod, a square obelisk, a 45° pyramid or a 30° pyramid.

11. Method, according to any one of claims 1 to 10, characterized in that the filleted base (32, 106) is positioned at the intersection of the support layer (20, 26, Petition 870260056666, dated 11 / 06 / 2026, page 66 / 131 4 / 8 34, 108) and the rod (22, 28, 104).

12. Microneedle arrangement (50, 100, 150) formed using a single pull mold, characterized in that it comprises: a support layer (20, 26, 34, 108); a plurality of microneedles (10, 12, 14, 16, 52); and at least one bioactive material combined with a first dissolvable material to form a biodegradable matrix, wherein the plurality of microneedles (10, 12, 14, 16, 52) each has a shaft (22, 28, 104), a microneedle tip (24, 102), a threaded base (32, 106), and at least one relief feature (104), and wherein the support layer (34, 108) is formed from a conformable material.

13. Microneedle arrangement (50, 100, 150), according to claim 12, characterized in that the embossed feature is located at the intersection of the shaft and the tip of the microneedle.

14. Microneedle arrangement (50, 100, 150), according to claim 12 or 13, characterized in that the rod (22, 28, 104) is formed from the first soluble material or is formed from an insoluble material.

15. Microneedle arrangement (50, 100, 150), according to any one of claims 12 to 14, characterized in that the plurality of microneedles (10, 12, 14, 16, 52) each comprises a second dissolvable material that forms a fast-dissolving layer (30) on a portion of the shaft (22, 28, 104) that is adjacent to the microneedle tip (24, 102).

16. Microneedle arrangement (50, 100, 150), according to claim 15, characterized in that the second dissolvable material of the dissolution layer (30) is selected from a material that dissolves more rapidly than the biodegradable matrix containing the bioactive material.

17. Microneedle arrangement (50, 100, 150), according to any one of claims 12 to 16, characterized in that the support layer is formed from soluble material or from an insoluble material.

18. Microneedle arrangement (50, 100, 150), according to any one of claims 12 to 17, characterized in that the first dissolvable material comprises carboxymethylcellulose, trehalose, polyvinylpyrrolidone, maltodextrin, silk, hyaluronic acid, poly(lactic-co-glycolic acid), poly(lactic acid), poly(vinyl alcohol), polyethylene glycol or a combination thereof.

19. Microneedle arrangement (50, 100, 150), according to claim 15 or 16, characterized in that the second dissolvable material comprises a small molecular weight polymer, a fast-dissolving polymer, optionally wherein the second dissolvable material is glucose, trehalose, sucrose, polyvinylpyrrolidone, maltodextrin or a combination thereof.

20. Microneedle arrangement (50, 100, 150), according to claim 12, characterized in that the conformable material of the support layer (34) is an insoluble polymer that can bend to conform to the non-uniform topography of the skin.

21. Microneedle arrangement (50, 100, 150), according to any one of claims 12 to 20, characterized in that at least one bioactive material comprises two or more bioactive materials.

22. Microneedle arrangement (50, 100, 150), according to any one of claims 12 to 21, characterized in that one or more bioactive materials comprise cosmetics, Petition 870260056666, dated 11 / 06 / 2026, p. 68 / 131 6 / 8 dermal fillers, statins, growth factors, analgesics, antihistamines, vitamins, anesthetics, anti-aging agents, small molecule drugs, haptens, allergens, anti-inflammatory agents, proteins, peptides, microvesicles, exosomes, polyplexes (siRNA, shRNA, DNA vector complexes), recombinant viral vectors (i.e., Adenovirus, Lentivirus, Herpes Simplex Virus, Vaccinia Virus, Adeno-associated Viruses and their different serotypes), monoclonal and polyclonal antibodies and live or lysed cells.

23. Microneedle arrangement (50, 100, 150), according to any one of claims 12 to 22, characterized in that the plurality of microneedles (10, 12, 14, 16, 52) has a shape that includes a circular obelisk, a square obelisk, a chamfered obelisk, a pyramid on a square rod, an arrowhead shape on a rod, a pyramid head on a square obelisk rod, a conical head on a circular obelisk rod, a square obelisk, a 45° pyramid, or a 30° pyramid.

24. Microneedle arrangement (50, 100, 150), according to any one of claims 12 to 23, characterized in that the threaded base (32, 106) is positioned at the intersection of the support layer (20, 26, 34, 108) and the shaft (22, 28, 104).

25. Method for forming a mold, characterized in that it comprises: generating a 3D CAD drawing (132) of a microneedle arrangement (100) that includes a plurality of microneedles (10, 12, 14, 16, 52) with at least one relief feature (104); forming a master microneedle arrangement (134) using 3D CAD drawing (132); forming at least one replica (140) of the arrangement of Petition 870260056666, dated 11 / 06 / 2026, p.69 / 131 7 / 8 master microneedles (134); and form a production mold (18, 144) of the microneedle arrangement (100) using at least one replica (140), the production mold (18, 144) comprising a plurality of cavities, with each of the pluralities of cavities being molded to define a plurality of respective microneedles (10, 12, 14, 16, 52) each having a shaft (22, 28, 104), a microneedle tip (24, 102), a threaded base (32, 106), and at least one relief feature (104), wherein the production mold (18, 144) is formed from a flexible material.

26. Method according to claim 25, characterized in that the flexible material has sufficient elasticity to allow a molded microneedle arrangement (50, 100, 150) to be removed from the production mold (18, 144) in a single pull, without damaging the shape integrity of the plurality of microneedles (10, 12, 14, 16, 52), as defined by the production mold (18, 144).

27. Method according to claim 25 or 26, characterized in that at least one replica (140) includes a plurality of replicas (140), and the forming of the production mold (18, 144) comprises forming a microneedle arrangement holder (142) and combining the plurality of replicas (140) in the microneedle arrangement holder (142).

28. Method, according to any one of claims 25 to 27, characterized in that the support of the microneedle arrangement (142) is formed of a resin material.

29. Method according to claim 1, characterized in that it further comprises: coating at least one part of the plurality of Petition 870260056666, dated 11 / 06 / 2026, p. 70 / 131 8 / 8 microneedles (10, 12, 14, 16, 52) with a bioactive component.

30. Method according to claim 29, characterized in that the microneedle (10, 12, 14, 16, 52) is coated with the bioactive component by immersion coating, air spraying or a combination thereof. Petition 870260056666, dated 11 / 06 / 2026, p. 71 / 131

Images