Microneedle arrays for cancer therapy applications

Abstract

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

Description

[Technical field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of U.S. Provisional Application No. 62 / 076,385, filed November 6, 2014, the contents of which are incorporated herein by reference in their entirety.

[0002] Field The present disclosure relates to systems and methods for transdermal drug delivery, and in particular to systems and methods for making and using dissolvable microneedle arrays.

[0003] Government support acknowledged This invention was made with Government support under Grant Nos. EB012776, AI076060, and CA121973 awarded by the National Institutes of Health. The Government has certain rights in this invention. [Background technology]

[0004] background The distinct physical barrier function of skin poses a significant challenge to transdermal drug delivery. To address this challenge, various microneedle array-based drug delivery devices have been developed. For example, one conventional method uses solid or hollow microneedle arrays that do not contain any active ingredients. Such microneedle arrays can pretreat the skin by perforating the stratum corneum and upper layers of the epidermis to enhance transdermal drug penetration before topical application of biologic carriers or traditional patches. This method has been shown to significantly increase the permeability of the skin, but it offers only limited ability to control the dosage and amount of drug or vaccine delivered.

[0005] Conventional methods using surface-coated solid microneedles or hollow microneedles attached to biologic reservoirs have significant limitations, including limitations on drug-dose combinations and in some cases complex fabrication procedures and special application settings. Although some solid microneedle arrays that are biodegradable and dissolvable have been developed, the use of these systems has been limited by fabrication processes that are limited in the active ingredients that can be embedded in the array in an effective and efficient manner. Summary of the Invention [Problem to be solved by the invention]

[0006] Thus, although transdermal delivery of biologics using microneedle array-based devices offers compelling theoretical advantages over prevalent oral and needle-based drug delivery methods, significant practical limitations exist in the design and fabrication associated with microneedle arrays constructed using conventional processes. [Means for solving the problem]

[0007] Abstract The systems and methods disclosed herein include a dissolvable microneedle array-based skin delivery platform that can provide efficient, precise and reproducible delivery of biologically active molecules to human skin. The microneedle array delivery platform can be used to deliver a wide range of bioactive ingredients to patients, including chemotherapeutic agents as described herein for cancer treatment applications.

[0008] The foregoing and other objects, features and advantages of the disclosed embodiments will become more apparent from the following detailed description which proceeds with reference to the accompanying drawings. In an embodiment of the present invention, for example, the following items are provided: (Item 1) 1. A dissolvable microneedle array for percutaneous insertion into a patient, comprising: a therapeutically effective amount of one or more bioactive ingredients, including at least one chemotherapeutic agent; Base, and A plurality of microneedles extending from the base. Including, A microneedle array, wherein substantially all of the one or more bioactive components are located within the plurality of microneedles such that the base is formed substantially without any bioactive components contained therein. (Item 2) 2. The microneedle array of item 1, wherein the one or more bioactive components include doxorubicin. (Item 3) 3. The microneedle array of item 2, wherein the amount of doxorubicin is about 50 to 200 micrograms. (Item 4) 2. The microneedle array of item 1, wherein the one or more bioactive components include at least two different chemotherapeutic agents. (Item 5) 5. The microneedle array of item 4, wherein the two different chemotherapeutic agents comprise doxorubicin and at least one other anthracycline drug. (Item 6) 2. The microneedle array of item 1, wherein the at least one chemotherapeutic agent comprises a cytotoxic agent and an immunostimulant. (Item 7) 7. The microneedle array of item 6, wherein the immunostimulant comprises at least one adjuvant. (Item 8) 7. The microneedle array of item 6, wherein the immunostimulant comprises at least one TLR antagonist. (Item 9) 7. The microneedle array of item 6, wherein the immunostimulatory agent comprises at least one ribonucleotide or deoxyribonucleotide. (Item 10) 7. The microneedle array of item 6, wherein the immunostimulatory agent comprises at least one type of dsRNA. (Item 11) 7. The microneedle array of item 6, wherein the immunostimulatory agent comprises at least one poly(I:C) derivative. (Item 12) 7. The microneedle array of item 6, wherein the immunostimulatory agent comprises poly(I:C). (Item 13) 7. The microneedle array of item 6, wherein the immunostimulatory agent comprises poly-ICLC. (Item 14) 7. The microneedle array of item 6, wherein the cytotoxic agent is doxorubicin and the immunostimulatory agent comprises poly(I:C). (Item 15) 7. The microneedle array of claim 6, wherein the cytotoxic agent is doxorubicin and the immunostimulatory agent comprises poly-ICLC. (Item 16) 2. The microneedle array of claim 1, wherein the one or more bioactive components are locally concentrated within the plurality of microneedles such that the one or more bioactive components are generally present only in the top half of each microneedle of the microneedle array. (Item 17) 2. The microneedle array of claim 1, wherein the plurality of microneedles are preformed to have a shape including a first cross-sectional dimension at a top portion, a second cross-sectional dimension at a bottom portion, and a third cross-sectional dimension at a middle portion, the middle portion being located between the top portion and the bottom portion, and the third cross-sectional dimension being greater than the first and second cross-sectional dimensions. (Item 18) 18. The microneedle array of claim 17, wherein the one or more bioactive ingredients are substantially concentrated in a region at or above the middle portion. (Item 19) Item 19. The microneedle array of item 18, wherein each microneedle generally tapers to a point above the midsection and each microneedle generally tapers to a smaller cross-sectional dimension below the midsection. (Item 20) 2. The microneedle array of item 1, wherein each microneedle comprises multiple layers of dissolvable biocompatible material. (Item 21) 21. The microneedle array of claim 20, wherein the dissolvable biocompatible material is carboxymethylcellulose. (Item 22) 21. The microneedle array of item 20, wherein the one or more bioactive components comprise at least two different chemotherapeutic agents, the at least two different chemotherapeutic agents comprising doxorubicin and at least one other anthracycline drug. (Item 23) 5. The microneedle array of item 4, wherein the at least two chemotherapeutic agents are selected from the group consisting of chemotherapeutic agents, adjuvants, and chemoattractants for cancer chemoimmunotherapy applications. (Item 24) 1. A method of fabricating a microneedle array by forming a sheet of material having a plurality of layers, comprising: applying a first solution of a soluble biocompatible material having one or more bioactive components therein to a microneedle array fabrication mold, the one or more bioactive components comprising at least one chemotherapeutic agent in a therapeutically effective amount; applying a second solution of a soluble biocompatible material that does not contain one or more active ingredients to the microneedle array mold; and drying the first and second solutions to form a solid microneedle array comprising a base and a plurality of microneedles extending from the base; The one or more active ingredients are substantially concentrated within the plurality of microneedles. (Item 25) 25. The method of claim 24, wherein the one or more biologically active ingredients comprise doxorubicin. (Item 26) The method according to item 25, wherein the amount of doxorubicin is about 50 to 200 micrograms. 25. The method of claim 24, wherein the one or more bioactive ingredients comprise at least two different chemotherapeutic agents. (Item 28) 28. The method of claim 27, wherein the at least two different chemotherapeutic agents include doxorubicin and at least one other anthracycline drug, and the dissolvable biocompatible material is carboxymethylcellulose. (Item 29) 25. The method of claim 24, wherein the at least one chemotherapeutic agent comprises both a cytotoxic agent and an immunostimulatory agent. (Item 30) 30. The method of claim 29, wherein the immunostimulant comprises at least one adjuvant. (Item 31) 30. The method of claim 29, wherein the immunostimulant comprises at least one TLR antagonist. (Item 32) 30. The method of claim 29, wherein the immunostimulatory agent comprises at least one ribonucleotide or deoxyribonucleotide. (Item 33) 30. The method of claim 29, wherein the immunostimulatory agent comprises at least one dsRNA. (Item 34) 30. The method of claim 29, wherein the immunostimulatory agent comprises at least one poly(I:C) derivative. (Item 35) 30. The method of claim 29, wherein the immunostimulatory agent comprises poly(I:C). (Item 36) 30. The method of claim 29, wherein the immunostimulatory agent comprises poly-ICLC. (Item 37) 30. The method of claim 29, wherein the cytotoxic agent is doxorubicin and the immunostimulatory agent comprises poly(I:C). (Item 38) 30. The method of claim 29, wherein the cytotoxic agent is doxorubicin and the immunostimulatory agent comprises poly-ICLC. (Item 39) 25. The method of claim 24, wherein the one or more bioactive ingredients are substantially concentrated in the top half of each microneedle of the microneedle array. (Item 40) 25. The method of claim 24, further comprising removing portions from the sheet of material until a microneedle array is formed having a base and a plurality of microneedles extending from the base, The method, wherein the removing of portions from the sheet array includes forming the microneedle array such that the one or more bioactive components are concentrated within each microneedle of the microneedle array and the one or more bioactive components are substantially absent from the base. (Item 41) Item 41. The method of item 40, wherein the method of removing portions comprises micromilling the sheet of material to form a microneedle array. (Item 42) 1. A method of treating skin cancer, comprising: selecting a subject having or at risk of having skin cancer; and Applying the microneedle array according to item 1 to an area of ​​the subject to deliver an effective amount of the at least one chemotherapeutic agent to the subject. method. (Item 43) 43. The method of claim 42, wherein the one or more bioactive components of the microneedle array comprise doxorubicin. (Item 44) The method according to Item 43, wherein the amount of doxorubicin is about 50 to 200 micrograms. Item 44. The method of item 43, wherein the one or more bioactive components of the microneedle array comprise at least two different chemotherapeutic agents. (Item 46) Item 47. The method of item 42, wherein the microneedle array comprises a dissolvable biocompatible material. 47. The method of claim 46, wherein the dissolvable biocompatible material is carboxymethylcellulose. (Item 48) 46. ​​The method of claim 45, wherein the at least two different chemotherapeutic agents comprise doxorubicin and at least one other anthracycline drug, and the microneedle array comprises a dissolvable biocompatible material. (Item 49) 46. ​​The method of claim 45, wherein the at least two chemotherapeutic agents include both a cytotoxic agent and an immunostimulatory agent. (Item 50) 50. The method of claim 49, wherein the immunostimulant comprises at least one adjuvant. (Item 51) 50. The method of claim 49, wherein the immunostimulant comprises at least one TLR antagonist. (Item 52) 50. The method of claim 49, wherein the immunostimulatory agent comprises at least one ribonucleotide or deoxyribonucleotide. (Item 53) 50. The method of claim 49, wherein the immunostimulatory agent comprises at least one dsRNA. (Item 54) 50. The method of claim 49, wherein the immunostimulatory agent comprises at least one poly(I:C) derivative. (Item 55) 50. The method of claim 49, wherein the immunostimulatory agent comprises poly(I:C). (Item 56) 50. The method of claim 49, wherein the immunostimulatory agent comprises poly-ICLC. (Item 57) 50. The method of claim 49, wherein the cytotoxic agent is doxorubicin and the immunostimulatory agent comprises poly(I:C). (Item 58) 50. The method of claim 49, wherein the cytotoxic agent is doxorubicin and the immunostimulatory agent comprises poly-ICLC. [Brief description of the drawings]

[0009] [Figure 1]FIG. 1 is a diagram illustrating exemplary microneedles and their dimensions.

[0010] [Diagram 2] FIG. 2 is a diagram illustrating an exemplary microneedle array and its dimensions.

[0011] [Diagram 3] 3A and 3B show exemplary microneedles tipped with an active ingredient.

[0012] [Figure 4] 4A and 4B show exemplary microneedles tipped with an active ingredient.

[0013] [Diagram 5] 5A and 5B show exemplary microneedles tipped with an active ingredient.

[0014] [Figure 6] 6A and 6B show exemplary microneedles tipped with an active ingredient.

[0015] [Figure 7] FIG. 7 shows a diagram of the miniature precision micromilling system used to fabricate the microneedle master mold.

[0016] [Figure 8] FIG. 8 is an SEM image of a micromilled master mold with pyramidal needles.

[0017] [Figure 9] FIG. 9 is an SEM image of the pyramid production mold.

[0018] [Figure 10]FIG. 10 is an SEM image of a magnified segment of the production mold, showing the pyramidal needle molding well in the center of the image.

[0019] [Figure 11] 11A-11D show exemplary CMC solids and embedded active ingredients.

[0020] [Figure 12] 12A and 12B show an exemplary CMC solid and an embedded active ingredient.

[0021] [Figure 13] FIG. 13 is a schematic diagram of an exemplary vertical multi-layer stack structure and a method for fabricating the same.

[0022] [Figure 14] FIG. 14 is a schematic diagram of an exemplary microneedle array fabricated using the layering and spatial distribution technique of an embedded active ingredient.

[0023] [Figure 15] FIG. 15 is a schematic diagram of an exemplary microneedle array fabricated in a spatially controlled manner.

[0024] [Figure 16A] FIG. 16A is an SEM image of multiple pyramidal shaped microneedles.

[0025] [Figure 16B] FIG. 16B is an SEM image of a single pyramidal shaped microneedle.

[0026] [Figure 17] FIG. 17 is an SEM image of a pillar-shaped molded microneedle.

[0027] [Figure 18] FIG. 18 is a micrograph of pyramidal molded microneedles.

[0028] [Figure 19] FIG. 19 is a micrograph of a pillar-shaped molded microneedle.

[0029] [Figure 20] FIG. 20 illustrates a variety of microneedle geometries that can be formed using a micromilled master mold or by directly micromilling a block of material.

[0030] [Figure 21] FIG. 21 shows a test apparatus for performing the destructive and piercing tests.

[0031] [Figure 22] FIG. 22 shows the force-displacement curves of a pillar-shaped microneedle (left) and a pyramidal microneedle (right).

[0032] [Diagram 23] FIG. 23 shows finite element models of microneedle deflection for a pillar-shaped microneedle (left) and a pyramidal microneedle (right).

[0033] [Figure 24] FIG. 24 shows various stereomicrographs of pyramidal (A, C, E) and pillar (B, D, F) shaped microneedle penetration in skin explants.

[0034] [Diagram 25] 25A, 25B, and 25C show the effectiveness of the microneedle array in penetrating skin explants.

[0035] [Figure 26] 26A and 26B show in vivo delivery of particulates to skin-draining lymph nodes of microneedle array-immunized mice.

[0036] [Figure 27] FIG. 27 is a bar graph showing the immunogenicity of microneedle-delivered model antigens.

[0037] [Figure 28] FIG. 28 is a bar graph showing the stability of active cargo on CMC microneedle arrays upon storage.

[0038] [Figure 29] 29A and 29B show induction of apoptosis in epidermal cells delivered with Cytoxan® (cyclophosphamide) by a microneedle array.

[0039] [Diagram 30] FIG. 30 illustrates microneedle geometries that can be formed by directly micromilling a block of material.

[0040] [Diagram 31] FIG. 31 is a stereo microscope image of a directly fabricated solid-state CMC microneedle array.

[0041] [Diagram 32] FIG. 32 is a stereo microscope image of a portion of the microneedle array of FIG.

[0042] [Diagram 33] FIG. 33 is a schematic cross-sectional view of a mold assembly for making blocks or sheets of material for direct micromilling.

[0043] [Diagram 34] FIG. 34 is a schematic cross-sectional view of a drying apparatus that can be used to dry blocks or sheets of material for direct micromilling.

[0044] [Diagram 35]FIG. 35: Flow cytometry analysis of GFP-expressing target 293T cells.

[0045] [Diagram 36] FIG. 36 shows the stability of microneedle-embedded virus after several days of storage.

[0046] [Figure 37] FIG. 37 shows expression and immunogenicity of adenovectors delivered with microneedle arrays.

[0047] [Figure 38] FIG. 38 illustrates an applicator for inserting microneedles into target tissue.

[0048] [Figure 39] FIG. 39 illustrates an applicator head design for use with the applicator shown in FIG.

[0049] [Diagram 40] FIG. 40 is a schematic diagram of the dimensional movement of the applicator head.

[0050] [Figure 41A] FIG. 41A compares tumor growth over time for MNA-delivered chemoimmunotherapy of B16 melanoma, including control subjects who did not receive any treatment, as well as subjects treated with doxorubicin incorporated into the same MNA.

[0051] [Figure 41B] FIG. 41B compares tumor growth over time for MNA-delivered chemoimmunotherapy of B16 melanoma, including control subjects who received no treatment, as well as subjects treated with poly(I:C) incorporated into the same MNA.

[0052] [Figure 41C]FIG. 41C compares tumor growth over time for MNA-delivered chemoimmunotherapy of B16 melanoma, including control subjects who received no treatment, and subjects treated with both doxorubicin and poly(I:C) incorporated into the same MNA.

[0053] [Figure 41D] FIG. 41D compares survival over time between subjects who received no treatment, doxorubicin mediated by MNA, poly(I:C) mediated by MNA, and doxorubicin and poly(I:C) incorporated into the same MNA.

[0054] [Diagram 42] FIG. 42 illustrates representative images on day 5 comparing control animals and animals treated with MNA-delivered chemoimmunotherapy of B16 melanoma.

[0055] [Diagram 43] FIG. 43 illustrates representative images at day 24 comparing control animals and animals treated with MNA-delivered chemoimmunotherapy of B16 melanoma.

[0056] [Diagram 44] FIG. 44 demonstrates a reduction in lung lesions in mice re-challenged after the initial MNA-delivered chemo-immunotherapy treatment.

[0057] [Diagram 45] FIG. 45 demonstrates lung lesions in challenged mice that did not receive the initial MNA-delivered chemo-immunotherapy treatment.

[0058] [Diagram 46] FIG. 46 compares the number of lung tumor foci in untreated mice and mice treated with MNA-delivered chemoimmunotherapy. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0059] Detailed Description The following description is exemplary in nature and is in no way intended to limit the scope, applicability, or configuration of the disclosed embodiments. Various changes to the described embodiments can be made in the function and arrangement of elements described herein without departing from the scope of the disclosure.

[0060] As used in this application and claims, the singular forms "a," "an," and "the" include the plural unless the context clearly requires otherwise. Additionally, the term "includes" means "comprises." As used herein, the term "biological agent," "active ingredient," "bioactive ingredient," "bioactive material," or "cargo" refers to a medicamentously active agent, such as analgesics, anesthetics, antiasthmatics, antibiotics, antidepressants, antidiabetic agents, antifungals, antihypertensives, antiinflammatory agents, antineoplastic agents, anxiolytics, enzymatically active agents, nucleic acid constructs, immunostimulants, immunosuppressants, vaccines, and the like. Bioactive materials may include soluble materials, insoluble but dispersible materials, natural or formulated macro-, micro-, and nanoparticulates, and / or mixtures of two or more of soluble materials, dispersible insoluble materials, and natural and / or formulated macro-, micro-, and nanoparticulates.

[0061] As used herein, the term "chemotherapeutic agent" refers to any chemical agent that has therapeutic utility in treating diseases characterized by abnormal cell proliferation. Such diseases include tumors, neoplasms, and cancers. Chemotherapeutic agents include those known to those skilled in the art for treating tumors, such as melanoma, including doxorubicin. Chemotherapeutic agents also include immunostimulants (specific and non-specific) known to those skilled in the art to stimulate the immune system to reject and destroy tumors, such as poly(I:C) and poly-ICLC.

[0062] In one example, the chemotherapeutic agent is administered to a subject to prevent or treat skin cancer. "Skin cancer" as used herein refers to a malignant growth on the skin, which may have many causes. Skin cancer generally develops in the epidermis (the outermost layer of the skin), and thus the tumor is usually clearly visible. Skin cancer includes both melanoma and non-melanoma types of cancer. Melanoma (also known as malignant melanoma or cutaneous melanoma) is a cancer that begins in melanocytes. Melanoma tumors are usually brown or black, since most melanoma cells still produce melanin. Non-melanoma skin cancer includes all skin cancers except malignant melanoma. Thus, for example, melanoma chemotherapeutic agents include agents that prevent or inhibit the development or metastasis of melanoma.

[0063] An amount of an agent that is sufficient to produce a desired response, such as reducing or inhibiting one or more signs or symptoms associated with a condition or disease. A dosage that achieves a target tissue concentration when administered to a subject is generally used. In some examples, an "effective amount" is one that treats one or more symptoms and / or underlying causes of any of the disorders or diseases. In some examples, an "effective amount" is a therapeutically effective amount of an agent alone or with additional therapeutic agent(s) that induces a desired response, such as preventing progression, slowing progression, or causing regression of skin cancer.

[0064] As used herein, the term "preformed" means that a structure or element is made, constructed and / or formed into a particular shape or configuration prior to use. Thus, a preformed shape or configuration of a microneedle array is the shape or configuration of the microneedle array prior to insertion of one or more of the microneedles of the microneedle array into a patient.

[0065] Although the operations of exemplary embodiments of the disclosed methods may be described in a particular sequential order for convenient presentation, it should be understood that the disclosed embodiments may encompass orders of operations other than the particular sequential order disclosed. For example, operations described as sequential may in some cases be rearranged or performed simultaneously. Furthermore, descriptions and disclosures presented in connection with one particular embodiment are not limited to that embodiment and may apply to any embodiment disclosed.

[0066] Moreover, for the sake of brevity, the accompanying drawings may not show the various ways in which the disclosed systems, methods and devices can be used in combination with other systems, methods and devices that are readily discernible by those of ordinary skill in the art based on this disclosure. Furthermore, this description occasionally uses terms such as "generate" and "provide" to describe the disclosed methods. These terms are high-level abstractions of actual operations that can be performed. The actual operations that correspond to these terms may vary depending on the particular implementation and are readily discernible by those of ordinary skill in the art based on this disclosure.

[0067] Tip-loaded microneedle array Dissolvable microneedle arrays allow for efficient and safe delivery of drugs and vaccines to skin and mucosal surfaces. However, the homogeneous nature of conventional microneedle array fabrication can result in inefficient drug delivery. Although the drug or other cargo to be delivered to the patient is generally incorporated throughout the microneedle array matrix, in reality, only the microneedles enter the skin, and therefore only the cargo contained within the volume of the individual needles is deliverable. Thus, the majority of the drug or other cargo that is localized in the non-needle components (e.g., the support structure of the array) is never delivered to the patient and is generally discarded as waste.

[0068] 1 and 2 show exemplary dimensions of microneedles and microneedle arrays. Based on the exemplary sizes shown in FIGS. 1 and 2, a microneedle array with an active ingredient homogenously distributed throughout the array exhibits greater than 40 percent active ingredient waste. For example, an array having a total area of ​​61 mm 2 and the area of ​​the microneedle array is 36 mm 2 1 and 2 show a particular size array and shape of microneedles, it should be understood that similar waste products will be present in any other size microneedle array where the active ingredient is homogenously distributed throughout the array, regardless of the size of the array or the shape of the microneedles involved.

[0069] The systems and methods described herein provide a novel microneedle array fabrication technique that utilizes fully dissolvable microneedle array substrates and unique microneedle geometries that enable the effective delivery of a wide range of active ingredients, including a wide range of protein and / or small molecule pharmaceuticals and vaccines.

[0070] As described in more detail herein, in some embodiments, the technology can also uniquely enable simultaneous co-delivery of multiple chemically distinct agents for multifunctional drug delivery. Examples of the utility of these devices include, for example, (1) simultaneous delivery of multiple antigens and adjuvants to generate polyvalent immune responses associated with infectious disease prevention and cancer therapy, (2) co-delivery of chemotherapeutic agents, immunostimulants, adjuvants and antigens to enable simultaneous adjuvant tumor therapy, and (3) localized dermal delivery of multiple therapeutic agents without systemic exposure to treat a wide variety of skin diseases.

[0071] In some embodiments, the systems and methods disclosed herein relate to novel fabrication techniques that allow various active ingredients to be incorporated into the needle tips. Thus, by localizing the active ingredients in this way, the remainder of the microneedle array volume can be prepared using less expensive matrix materials that are inactive and generally considered safe. The end result is (1) a reduction in the waste of non-deliverable active ingredients incorporated into the non-needle portion of the microneedle array, and (2) a greatly improved efficiency of drug delivery based on a higher drug concentration in the needle tips that penetrate the skin. This technological advance dramatically improves the economic feasibility relative to the cost of the drug cargo and increases the effective cargo delivery capacity per needle of these novel microneedle arrays.

[0072] Figures 3A, 3B, 4A and 4B show various embodiments of microneedle arrays in which the active ingredient is concentrated at the tips of the microneedles of each array. Thus, in contrast to conventional microneedle arrays, the active ingredient is not present in a uniform concentration throughout the microneedle array, since there is little or no active ingredient present on the supporting base structure. Furthermore, in some embodiments (e.g., as shown in Figures 3A, 3B, 4A and 4B), not only is there little or no active ingredient present on the supporting structure, but the location of the active ingredient is concentrated in the top half of each microneedle of the array.

[0073] 5A and 5B show exemplary images of microneedles of a microneedle array containing an active ingredient concentrated in the top half of each individual microneedle. The active ingredient is shown as fluorescent particles concentrated at the tip of the microneedle, which is defined by the extent of the microneedle extending from the base in a narrowing and / or tapering manner. The base further extends from the support structure of the array.

[0074] Figures 6A and 6B show additional exemplary images of microneedles of a microneedle array containing an active ingredient concentrated in the top half of each individual microneedle. In Figure 6A, the active ingredient is concentrated at the tip of the microneedle and is BSA-FITC. In Figure 6B, the active ingredient is also concentrated at the tip of the microneedle and is OVA-FITC.

[0075] As noted above, in some embodiments, individual microneedles may contain the active ingredient only in the top half of the microneedle. In other embodiments, individual microneedles may contain the active ingredient only in the narrowed portion at or near the tip of the microneedle. In yet other embodiments, individual needles may contain the active ingredient throughout the portion of the microneedle that extends from the support structure.

[0076] In the following embodiments, various exemplary methods are described for fabricating microneedle arrays that contain one or more active ingredients concentrated in the top half and / or tips of the microneedles of each microneedle array.

[0077] Microneedle arrays fabricated by sequential micromolding and spin-drying techniques The following steps describe an exemplary method of fabricating a microneedle array using sequential micromolding and spin drying. The active ingredient / cargo can be prepared in a compatible solvent at a desired useful concentration. As described herein, the solvent for the active ingredient(s) can be cargo-specific and can include a wide range of liquids, including, for example, water, organic polar and / or non-polar liquids. Examples of active ingredients are discussed in more detail below, as well as various information about these active ingredients, including the maximum loading capacity tested for various microneedle arrays.

[0078] If necessary, multiple loading cycles can be performed to achieve higher active cargo loadings if required for a particular application. Additionally, multiple active cargoes can be loaded as a single solution in a single loading cycle as a composite solution or in multiple cycles (e.g., repeated loading cycles as described below) depending on the specific cargo compatibility requirements of each individual cargo. Also, particulate cargoes (including those with nano- and micro-sized geometries) can be prepared as suspensions at the desired particle number / volume density. EXAMPLES

[0079] Example 1 a) In a micromilling embodiment, as described in more detail below, a working stock solution / suspension of active cargo is milled into a microsphere, for example, having a surface area of ​​1 cm 2 Approximately 40 μl per microneedle can be applied to the surface of a mold for producing a microneedle array.

[0080] b) To fill the needles of the microneedle array fabrication mold with the working cargo stock, the microneedle array fabrication mold containing the active cargo(s) can be centrifuged at 4500 rpm for 10 minutes.

[0081] c) Excess cargo solution / suspension can be removed and the surface of the microneedle array fabrication mold can be polished to a mold surface area of ​​1 cm 2 The cells can be washed with 100 μl of phosphate buffered saline (PBS) per well, or with the solvent used to prepare the working stock of the active cargo.

[0082] d) The microneedle array fabrication mold containing the active cargo stock solution / suspension in the needle cavities can be spin-dried at 3500 rpm for 30 minutes at the required temperature with a continuous flow of purge gas in the centrifuge at 0-50 L / min to facilitate concentration of the drying active cargo(s) at the needle tips. The purge gas can be introduced into the centrifuge chamber through a tubular inlet. The moisture content can be reduced using a dehumidifier regulated to the required temperature and recirculated into the centrifuge chamber. The purge gas can be air, nitrogen, carbon dioxide, or another inert or active gas as required for the particular cargo(s). The flow rate is measured by a flow meter and controlled by a circulation pump device.

[0083] e) Mold area for manufacturing microneedle array: 1 cm 2 100 μl of 20% CMC90 hydrogel in H2O can be added per well to the surface of a microneedle array fabrication mold to load the structural components of the microneedle array device.

[0084] f) The microneedle array fabrication mold can be centrifuged at 4500 rpm for 10 minutes at the required temperature without purge gas exchange in the centrifuge chamber to fill the needle cavities of the microneedle array fabrication mold with CMC90 hydrogel, followed by a 30 minute incubation period to allow rehydration of the active cargo(s) previously deposited on the microneedle array tips.

[0085] g) The microneedle array fabrication mold can be centrifuge at 3500 rpm for 3 hours or more at the required temperature with a constant purge gas flow of 0-50 L / min through the centrifuge chamber to spin dry the MNA device to a moisture content of less than 5%.

[0086] h) The dried microneedle array device can then be separated from the microneedle array fabrication mold and stored under desired conditions. In some embodiments, the CMC90-based device may be storable between about 50°C and -86°C.

[0087] Examples of fabricated microneedle arrays carrying active cargo at their tips can be seen in Figures 3A-6B.

[0088] Micromilled master molds and spin-molded microneedle arrays In the following embodiments, a micromilling step is performed to create microneedle arrays of various specifications, however, it should be understood that the following embodiments describe certain details of microneedle array fabrication that may be applicable to processes of microneedle array fabrication that do not involve a micromilling step, including the processes described above in the previous examples.

[0089] In the following embodiments, an apparatus and method for fabricating dissolvable microneedle arrays using master molds formed by micromilling techniques is described. For example, microneedle arrays can be fabricated based on a master mold (positive) - production mold (negative) - array (positive) methodology. Micromilling techniques can be used to generate a variety of microscale geometries in virtually any type of material, including metal, polymer, and ceramic parts. Micromilled master molds of various shapes and configurations can be effectively used to generate multiple identical female production molds. The female production molds can then be used to microcast a variety of microneedle arrays.

[0090] FIG. 7 shows an example of a precision micromilling system that can be used to fabricate the microneedle master mold. Mechanical micromilling uses microscale (e.g., as small as 10 μm) milling tools in a precision computer-controlled miniature machine tool platform. The system can include a microscope to view the surface of the workpiece being cut by the microtool. The microtool can be rotated at very high speeds (200,000 rpm) to cut the workpiece to create the desired shape. As mentioned above, the micromilling process can be used to create complex geometric features using many types of materials. For example, various types of tools can be used in the micromilling process, including carbide microtools. However, in a preferred embodiment, diamond tools can be used to fabricate the microneedle arrays on the master mold. Diamond tools can be preferred over other types of tools because they are harder than traditional materials such as carbides and can result in cleaner cuts on the surface of the workpiece.

[0091] The mastermold can be micromilled from a variety of materials, including, for example, Cirlex® (DuPont, Kapton® polyimide), which is the mastermold material described in the exemplary embodiment. The mastermold can be used to fabricate a flexible production mold from a suitable material, such as SYLGARD® 184 (Dow Corning), which is the production material described in the exemplary embodiment below. The mastermold is desirably formed from a material that can be reused, such that a single mastermold can be used repeatedly to fabricate multiple production molds. Similarly, each production mold is desirably capable of fabricating multiple microneedle arrays.

[0092] Mastermolds can be made relatively quickly using micromilling techniques. For example, a mastermold containing a 10 mm x 10 mm array with 100 microneedles can take less than 2 hours to micromill, and in some embodiments, less than about 30 minutes. Thus, the short ramp-up time allows different geometries to be rapidly fabricated, thereby enabling rapid development of microneedle arrays and facilitating experimentation and research of various microneedle parameters.

[0093] The master mold material is preferably capable of being cleanly separated from the production mold material and is preferably capable of withstanding any elevated cure temperatures that may be required to cure the production mold material. For example, in the illustrated embodiment, the silicone-based compound SYLGARD® 184 (Dow Corning) is the production mold material, which material typically requires a cure temperature of about 80-90 degrees Celsius.

[0094] The mastermold can be made in a variety of sizes. For example, in an exemplary embodiment, the mastermold was made of 1.8 mm thick Cirlex® (DuPont, Kapton® polyimide) and 5.0 mm thick acrylic sheets. Each sheet can be first flattened by a micromilling tool, and the locations where the microneedles will be made can be elevated from the rest of the surface. The microtool can be used in conjunction with a numerically controlled micromilling machine (FIG. 1) to create the features of the microneedles (e.g., defined by the mastermold). In that manner, the micromilling process can provide full control of the dimensions, sharpness, and spatial distribution of the microneedles.

[0095] FIG. 8 is an image from a scanning electron microscope (SEM) showing the structure of a micromilled master mold with multiple pyramidal needles. As shown in FIG. 8, a circular groove can be formed around the microneedle array of the master mold to generate an annular (e.g., circular) wall section in the production mold. The circular wall section of the production mold can facilitate the spin casting process discussed below. Although the wall section shown in FIG. 9 and the respective master mold structure shown in FIG. 8 are circular, it should be understood that wall sections or containment means of other geometric shapes can be provided. For example, the containment means can be formed in various shapes, including, for example, square, rectangular, trapezoidal, polygonal, or various irregular shapes, depending on which shape is desired for the microneedle array device.

[0096] As discussed above, the production mold can be made from SYLGARD® 184 (Dow Corning), which is a two-component transparent curable silicone elastomer that can be mixed in a 10:1 ratio of SYLGARD® to curing agent. This mixture can be degassed for about 10 minutes and applied to the master mold to form an approximately 8 mm layer, subsequently degassed again for about 30 minutes, and cured at 85° C. for 45 minutes. After cooling to room temperature, the master mold can be separated from the cured silicone, and the silicone production mold was trimmed to the edge of the circular wall section surrounding the array (FIG. 9). From a single master mold, a large number of production molds (e.g., 100 or more) can be produced with very little, if any, apparent degradation of the Cirlex® or acrylic master mold.

[0097] Figure 9 is an SEM image of a pyramidal production mold made as described above. Figure 10 shows a magnified segment of the production mold with a pyramidal needle molding well in the center of the image. The molding well is configured to receive a substrate (and any components added to the substrate) to form a microneedle having an outer shape defined by the molding well.

[0098] To construct a microneedle array, a substrate can be used to form portions of each microneedle that have and do not have a bioactive component. As discussed above, each microneedle may contain a bioactive component only in the microneedle, or in some embodiments, only in the top half of the microneedle, or in other embodiments, only in the portion of the microneedle that tapers near the tip. Thus, to control the delivery of the bioactive component(s) and to control the cost of the microneedle array, it is preferred that each microneedle has a portion that contains a bioactive component and a portion that does not contain a bioactive component. In the embodiments described herein, the portion that does not contain a bioactive component includes the support structure of the microneedle array, and in some embodiments, the base (e.g., bottom half) of each microneedle of the array.

[0099] A variety of materials can be used as the substrate for the microneedle array. Biodegradable solid microneedle structural substrates most commonly include poly(lactic-co-glycolic acid) (PLGA) or carboxymethylcellulose (CMC) based formulations, although other bases can also be used.

[0100] CMC is generally preferred over PLGA as the substrate for the microneedle array described herein. PLGA-based devices may be limited in drug delivery and vaccine applications due to the relatively high temperatures (e.g., 135 degrees Celsius or higher) and vacuum required for fabrication. In contrast, CMC-based matrices can be formed at room temperature with a simple spin-casting and drying process, making CMC microneedle arrays more desirable for the incorporation of sensitive biologics, peptides, proteins, nucleic acids, and various other bioactive components.

[0101] CMC hydrogels can be prepared from low viscosity sodium salt of CMC with or without active ingredients (as described below) in sterile dH2O. In an exemplary embodiment, CMC can be mixed with sterile distilled water (dH2O) and active ingredients to achieve a CMC concentration of about 25 wt%. The resulting mixture can be stirred until homogenous and equilibrated at about 4 degrees Celsius for 24 hours. During this period, CMC and any other ingredients can be hydrated and a hydrogel can be formed. The hydrogel can be degassed under vacuum for about 1 hour and centrifuged at about 20,000g for 1 hour to remove any residual micro-sized air bubbles that may interfere with the spin casting / drying process of the CMC microneedle array. The dry matter content of the hydrogel can be tested by drying a fraction of the hydrogel (10 g) at 85 degrees Celsius for about 72 hours. The ready-to-use CMC hydrogel is desirably stored at about 4 degrees Celsius until use.

[0102] Active ingredients can be incorporated into the CMC hydrogel at relatively high (20-30%) CMC-dry biologic weight ratios prior to the spin-casting process. Arrays are spin-cast at room temperature, making the process structurally compatible with the functional stability of a wide range of bioactive ingredients. Fabrication costs can be greatly reduced, as the master mold and production mold can be reusable for multiple fabrication cycles. The resulting dehydrated CMC microneedle arrays are generally stable at room temperature or slightly lower temperatures (such as about 4 degrees Celsius), preserving the activity of the incorporated biologic and facilitating easy low-cost storage and distribution.

[0103] In an exemplary embodiment, the surface of the production mold can be covered with about 50 μl of CMC hydrogel (for a mold with a diameter of 11 mm) and spin-cast by centrifugation at 2,500 g for about 5 minutes. After the initial CMC hydrogel layer, another 50 μl of CMC hydrogel can be layered on the mold and centrifuged at 2,500 g for about 4 hours. At the end of the drying process, the CMC microneedle array can be separated from the mold, trimmed from excess material at the edges, collected, and stored at about 4 degrees Celsius. The production mold can be cleaned and reused for further casting of microneedle arrays.

[0104] In some embodiments, the CMC solid can be formed with a layer that does not contain an active ingredient and a layer that contains an active ingredient. Figures 11A-11D show CMC solids with different shapes (Figures 11A and 11B) and embedded active cargo in the upper layer that becomes part of the microneedle containing the active ingredient after micromilling. Figure 11C shows micron-sized fluorescent particles layered on the surface of the non-active ingredient-containing layer, and Figure 11D shows an example of toluidine blue layered on the surface of the non-active ingredient-containing layer.

[0105] Figures 12A and 12B also show CMC solids with different shapes, with Figure 12B showing a square shape and Figure 12B showing a rectangular shape. Both CMC solids can be milled to dimensions for further processing as described herein. It should be understood that the geometries and active cargoes shown herein are not intended to be limiting to exemplary embodiments.

[0106] Example 2 CMC solids can be prepared with defined geometry and active cargo content in one or more layers of the prepared structure.Examples of active cargo integrated into CMC solids are described in more detail herein.After constructing the CMC solids with embedded active cargo contained in at least one layer of the CMC solids, the CMC solids can be milled and micromilled to project-specific dimensions to fabricate the microneedle device described herein.

[0107] Example 3 In another embodiment, one or more layers of active cargo can be embedded in a CMC solid for direct micromilling of microneedle arrays. Figure 13 shows a representative sample of vertical multilayer stacking and CMC embedding of active cargo on a CMC solid for direct micromilling of MNA devices.

[0108] In one exemplary method, microneedle arrays can be fabricated by preparing a CMC solid having a defined geometry and not containing any active cargo therein. The blank CMC solid can then be milled to the desired dimensions.

[0109] As shown in Figure 13, active cargo(s) can be deposited onto the CMC solid in a project-specific geometric pattern to specifically include the active cargo(s) at the tip of the micromilled MNA device.

[0110] Methods for depositing active cargo onto a CMC solid blank include, for example: 1) Direct printing using micronozzle-assisted droplet deposition; 2) Moving away from pre-printed matrices, 3) Droplet Deposition Using a Computer-Controlled Robotic System Examples include:

[0111] Figure 14 shows the layering and spatial distribution of embedded active cargo in a CMC solid block. After the first layer is deposited (A), it can be covered with a CMC layer (B), which provides a surface for subsequent deposition of active cargo (C). This process can be repeated (D-F) until all desired layers have been deposited and encased within a solid CMC block suitable for the micromilling process.

[0112] Figure 15 shows a schematic of a cross section of a CMC block encasing a deposit of active cargo in a spatially controlled manner (A). The method allows for three-dimensional control and placement of the active ingredient after micromilling in the MNA device (B). In panel (B) of Figure 15, placement of the active cargo is shown at the stem of the active cargo, but by control of the milling process, placement can be controlled vertically from the tip to the base of the microneedle. The colors represent different active ingredients, or different amounts / concentrations of the same material.

[0113] Thus, a method is provided for vertically layered deposition of active cargo on microneedles by sequentially depositing one or more active cargo on the surface of CMC solid, either in contact with each other or separated by a layer of CMC.In some embodiments, horizontal pattern deposition of active cargo can spatially separate cargo.By combining vertical and horizontal patterns of active cargo deposition, it is possible to realize three-dimensional delivery and distribution of each defined active ingredient, and further reduce the waste of active ingredient during the fabrication of microneedle array.

[0114] Microneedle-integrated adenovector The following embodiments are directed to dissolvable microneedle arrays such as those described herein, which incorporate infectious viral vectors into the dissolvable matrix of the microneedle array. Using this technology, for the first time, live viral vectors can be incorporated into the microneedle array. As described herein, the incorporation of viral vectors into the disclosed microneedle arrays stabilizes the viral vectors, so that they maintain their infectivity after incorporation and after long-term storage. The application of microneedle array-integrated adenovectors (MIA) to the skin transfects skin cells. In a vaccine context, the inventors have demonstrated that the application of MIA encoding HIV antigens to the skin results in a strong HIV-specific immune response. These results are described in detail in the following examples.

[0115] Example 4 The preparation method of microneedle-integrated adenovectors described herein preserves the viability of adenovirus particles during preparation and dry storage. These steps were specifically designed based on the physical and chemical properties of the CMC microneedle array. Viral viability in the CMC microneedle array is - by including low viscosity carboxymethylcellulose (CMC90) at a final concentration of 2.5% (step 2), and - timed and temperature-controlled spin-dry concentration of adenoviral particles at the tips of the microneedle array device (step 6), - achieved by controlled partial rehydration of the needle tip-loaded adenoviral particles (step 8).

[0116] Preparation of tip-loaded microneedle-integrated adenovectors (MIA): 1) Trehalose storage buffer (5% trehalose 2 × 10 in 20 mM Tris pH 7.8, 75 mM NaCl, 2 mM MgCl2, 0.025% Tween 80 (Sigma-Aldrich USA) 9Resuspend the adenoviral particles at a density of 100 particles / ml. 2) Mix the resuspended virus stock with an equal volume of 5% CMC90 prepared in trehalose storage buffer and dilute at 1 × 10 9 Result in an adenovirus working stock with a density of particles / ml. 3) The adenovirus working stock suspension is dispersed in a solution of 1 cm 2 40 μl per well is added to the surface of a mold for fabricating a microneedle array (as described in detail in other embodiments herein). 4) Centrifuge the mold at 4500 rpm for 10 minutes at 22° C. and fill the needle tip with adenovirus working stock. 5) Remove excess virus stock and pat dry the mold surface with a mold surface area of ​​1 cm. 2 Wash with 100 μl of phosphate buffered saline (PBS) solution per well. 6) The microneedle array mold containing the adenovirus stock solution only within the needle cavities is partially spin-dried at 3500 rpm for 10 minutes at 22°C. 7) Mold area 1cm 2 Add 100 μl of 20% structural non-cargo-containing CMC90 hydrogel in HO per well to the surface of the microneedle array mold to form the structure of the MIA device. 8) Centrifuge at 4500 rpm for 10 minutes at 22 °C to fill the needle cavity with 20% CMC90 and incubate for 30 minutes to rehydrate the adenovirus particles that were dried within the tip (steps 3-6, above). 9) Spin dry the MIA device to less than 5% moisture content by centrifugation at 3500 rpm for 3 hours at 22° C. with a constant air flow of 10 L / min through the centrifuge chamber. 10) Demold the dried MIA device and store at 4° C. or -80° C.

[0117] Example 5 We evaluated the efficacy and stability of MNA-incorporated recombinant adenovirus particles. Ad5.EGFP was incorporated into CMC hydrogel MNA for 10 min. 10A final product containing viral particles / MNA was produced. A control blank MNA was prepared exactly the same but without virus. Batches of Ad5.EGFP MNA and control MNA were stored at RT, 4°C, and -86°C and virus stability was assessed in an infection assay. The specific transduction activity of MNA-incorporated Ad5.EGFP virus was assessed in vitro using 293T cells. 2 × 10 6 Cells were plated at 1000 x g / well and transduced in duplicate with diluted virus suspensions, suspensions + empty MNAs (control), or Ad5.EGFP MNAs stored at RT, 4°C, and -86°C for the indicated times. Non-transduced wells were included as negative controls. Cell populations were first analyzed after 24 hours for GFP expression by flow cytometry (representative histograms are shown in Figure 35).

[0118] As shown in Figure 35, incorporating Ad5.EGFP into MNAs does not reduce transduction efficiency. Flow cytometry analysis of GFP-expressing target 293T cells 24 hours after transduction with identical titers of Ad5.EGFP in suspension or incorporated into CMC patches versus untransfected control cells. Figure 36 shows the stability of MNA-embedded Ad5.EGFP virus. GFP gene expression was assayed by flow cytometry as in Figure 37 and normalized to the infection efficiency of -86°C stored Ad5.EGFP suspensions.

[0119] The infection efficiency using the MNA Ad5.EGFP virus was found to be 87.92±4.5%, which is similar to that observed with traditional −86° C. stored Ad5.EGFP suspensions (FIG. 35 and FIG. 36), suggesting that the manufacturing process does not adversely affect the transduction efficiency of Ad-EGFP viral particles. To assess infectivity over time, the transfection efficiency of freshly prepared −86° C. stored Ad5.EGFP suspensions was compared to that of MNA-integrated Ad5.EGFP stored at RT, 4° C., or −86° C. for extended periods. Infectivity (normalized to Ad5.EGFP suspensions + empty CMC patches) over storage periods of up to 365 days is reported (FIG. 36). These results suggest that the infectivity of MNA Ad5.EGFP is remarkably stable upon storage at either 4° C. or −86° C., and somewhat stable at RT for up to 30 days.

[0120] These results demonstrate that microneedle array-delivered Ad transgenes are expressed in the skin and induce a strong cellular immune response. To specifically assess gene expression in vivo, we determined GFP expression in the skin following traditional intradermal injection (ID) or microneedle array-mediated intradermal delivery. We administered GFP to 10 mice either locally by ID injection or via a single microneedle array application. 8Ad5.GFP viral particles were delivered (Figure 37). Skin was harvested 48 hours later, cryosectioned, and counterstained with blue fluorescent DAPI to identify cell nuclei, then imaged by fluorescence microscopy. Significant cellular GFP expression was observed after both ID and microneedle array delivery. To assess immunogenicity, we assessed antigen-specific lytic activity in vivo after single ID or microneedle array immunization without boosting. To this end, we immunized groups of mice with codon-optimized SIVmac239 gag full-length, or E1 / E3 deleted Ad5-based vectors encoding SIVmac239 gag p17 antigen (Ad5.SIV gag, Ad5.SIV gag p17). Empty vector was used as a control (Ad5). We observed potent and similar levels of in vivo lytic activity specific for the dominant SIVgag p17-derived peptide KSLYNTVCV (SIVmac239 gag 76-84) following ID or microneedle array immunization with Ad5.SIV gag or Ad5.SIV gag p17 (Figure 37, CTL).

[0121] The microneedle array technology disclosed herein can also facilitate clinical gene therapy. It addresses, for example, at least two major limitations of conventional approaches. First, it allows for the stabilization and storage of recombinant viral vectors over extended periods of time. By rendering live viral vectors resistant to high and low temperatures with proven seroequivalence to frozen liquid formulations, microneedle array stabilization relieves the pressures associated with the "cold chain". Furthermore, integration into microneedle arrays allows for precise, consistent and reproducible dosing of viral vectors that is not feasible by conventional methods. Finally, viral vectors are repackaged into the only delivery device required, a biocompatible and fully disposable microneedle array that precisely directs delivery to the superficial layers of the skin.

[0122] Such gene delivery platforms are useful in bringing about patient-friendly clinical gene therapy. These microneedle arrays are engineered not to penetrate deep into vascular or neural structures, making gene delivery to human skin both painless and non-invasive. Furthermore, the fabrication process is flexible, allowing simple, rapid, low-cost production with efficient scale-up potential. Also, as the final product, the MIA device, it is stable at room temperature and inexpensive to transport and store. Combined, these structural and manufacturing advantages can enable broad, rapid clinical deployment, making this gene delivery technology readily applicable to the prevention and / or treatment of a wide range of human diseases. Furthermore, this approach can be extended to other vector-based vaccine platforms (e.g., vaccinia virus, AAV, etc.) that are currently restricted by the same limitations. For at least these reasons, the disclosed microneedle arrays and methods of using the same significantly advance the recombinant gene therapy field.

[0123] Microneedle Arrays - Exemplary Active Ingredients The various active ingredients are described in detail below. For convenience, the following examples are based on a microneedle array that is 6.3 x 6.3 mm. This size, and therefore cargo delivery, can be varied by increasing or decreasing by 2 to 100 fold.

[0124] General considerations for maximum active cargo amount include, for example, the total needle volume in the array and the solubility of the active ingredient(s) in the solvent (generally expected to be less than 50%). [Table 1] [Table 2] [Table 3]

[0125] Tip loading of live adenovirus generally involves the following modifications: a) Presence of 5% trehalose and 2.5% CMC90 in the tip-loading hydrogel suspension. b) The process temperature is maintained at 22°C.

[0126] Furthermore, lentiviral vectors generally require 4 °C processing and steam trap-based humidity control, and short epitope peptides are generally solubilized in DMSO, with a 4 h evaporation time for the solvent during tip loading.

[0127] The amount of the agent may vary depending on the particular application, and is generally selected to be in an amount sufficient to provide a therapeutic effect alone or in combination with other agents or materials. For example, in some embodiments, a therapeutically effective amount of doxorubicin may be about 25-300 micrograms, about 50-200 micrograms, or in other embodiments, about 75-125 micrograms. As used herein, "about" means plus or minus 5 percent of the amount. For example, "about 50-200 micrograms" includes 47.5-210 micrograms.

[0128] Microneedle Structure and Shape For each of the following embodiments, it should be understood that one or more layers of active ingredient may be provided in the microneedles of the microneedle arrays described above. Thus, for example, in some embodiments, the active ingredient is provided only within the microneedles that are not within the structural support of the array, such as those shown in Figure 15. Additionally, in other embodiments, the active ingredient is concentrated in the top half of the microneedles, such as the tips of the microneedles shown in Figures 3A-4B.

[0129] 16A and 16B are SEM images of a CMC microneedle array formed with multiple pyramidal projections (i.e., microneedles). The average tip diameter of the pyramidal needles shown in FIG. 16A is about 5-10 μm. As shown in FIG. 16B, the sides of the pyramidal needles can be formed with curved and / or arcuate surfaces that can facilitate insertion into the skin.

[0130] Figure 17 is another SEM image of a single needle of a microneedle array. The microneedle shown in Figure 17 is a base-extended pillar-type molded CMC microneedle. A base-extended pillar-type microneedle includes a base that is generally polygonal (e.g., rectangular) in cross section, and a protrusion extending from the base. The protrusion has a substantially rectangular lower portion and a tip that generally tapers to a point. The tip is generally pyramidal in shape, and the exposed surface of the pyramid can be flat or arcuate. The protrusion can be half or more of the total length of the needle.

[0131] Figures 18 and 19 show micrographs of pyramidal (Figure 18) and pillar-shaped (Figure 19) shaped CMC microneedles. Pyramid needles have a continuously increasing cross-sectional profile (dimension) from the needle tip to the needle base, so that as the needle enters the skin, the force required to continue pushing the pyramidal needle into the skin increases. In contrast, pillar-shaped needles generally have a continuous cross-sectional profile (dimension) once the generally rectangular portion of the protrusion is reached. Thus, pillar-shaped needles may be preferred over pyramidal needles, as they may allow for the introduction of the needle into the skin with less force.

[0132] Figure 20 shows schematics of microneedle shapes and structures that are generally suitable for fabrication by spin casting material onto a master mold formed by micromilling. The shapes and structures shown in Figure 20 do not contain any undercuts, so they generally do not interfere with the molding / demolding process. The structures in Figure 20 include (a) a roughly pyramidal microneedle, (b) a "sharp" pillar-type microneedle (without the base member of Figure 8), (c) a "wide" pillar-type microneedle, (d) a "short" pillar-type microneedle (having a short pillar section and a longer pointed section), and (e) a "filleted" pillar-type microneedle.

[0133] Although the volume of pyramidal microneedles may be larger than that of pillar-shaped microneedles, their increasing cross-sectional profile (dimension) requires increasing insertion force. Thus, the geometry of pyramidal microneedles can reduce the insertion depth and reduce the effective delivery volume. On the other hand, the smaller cross-sectional area and larger aspect ratio of pillar microneedles can lead to a lower breaking force limit. The smaller the apex angle α, the sharper the tip of the microneedle. However, if the apex angle is made too small (e.g., less than about 30 degrees), the resulting microneedle volume and mechanical strength can be reduced to undesirable levels.

[0134] The penetration force of the microneedle is inversely proportional to the sharpness of the microneedle, which is characterized not only by the included (apex) angle of the microneedle, but also by the radius of the microneedle tip. The apex angle is determined by the master mold geometry, while the sharpness of the tip also depends on the reliability of the mold. Micro-rimming the master mold described herein allows the precision of the mold geometry to be increased, which in turn increases the precision and reliability of the resulting production mold and the microneedle array formed by the production mold.

[0135] The increased precision of micromilling allows for more precise and precise elements to be included in the mold design. For example, as discussed in the next section below, forming fillets at the base of pillar-shaped microneedles can significantly increase the structural integrity of the microneedles, thereby reducing the likelihood that the microneedles will break or break when impacted with the skin. These fillets can significantly increase the strength of the microneedles, but do not interfere with the functional requirements of the microneedles (e.g., penetration depth and biologic volume). Such fillets are very small features that can be difficult to make in master molds formed by conventional techniques. However, the micromilling techniques described above allow for the inclusion of such small features with little or no difficulty.

[0136] Mechanical Integrity and Penetration Capability The microneedle array is preferably configured to penetrate the stratum corneum to deliver its cargo (e.g., a biologic or bioactive ingredient) to the epidermis and / or dermis while minimizing pain and bleeding by preventing penetration into deeper layers that may contain nerve endings and blood vessels. To evaluate the mechanical viability of the fabricated microneedle arrays, tests were performed on pyramidal and pillar-shaped microneedle arrays (e.g., as shown in Figures 7B and 8) as representative variations of array geometry. The first set of tests shows the fracture limit of the microneedles and involves pressing the microneedle array against a solid acrylic surface at a constant approach speed while simultaneously measuring the force and displacement until fracture occurs. The second set of tests shows the perforation ability of the microneedles against human skin explants.

[0137] Figure 21 shows the test setup designed for functional testing. The sample (i.e., the microneedle array) was mounted in a fixture, which was advanced toward a fixed acrylic artifact (PMMA surface) at a constant speed of approximately 10 mm / sec using a computer-controlled translation stage (ES14283-52 Aerotech, Inc.). A three-axis dynamometer (9256C1, Kistler, Inc.) received the acrylic artifact, allowing for sensitive measurement of force.

[0138] FIG. 22 shows the force-displacement curves of the data measured during the fracture test. The left curve represents the data obtained from the test of the pillar microneedle sample, and the right curve represents the data obtained from the test of the pyramidal microneedle. As can be seen in FIG. 22, the fracture of these two types of microneedles is significantly different, the pyramidal array undergoes plastic deformation (bending) while the pillar array exhibits the breakage of the pillar at its base. This different fracture behavior is coupled with considerably different displacement-force data. The fracture (fracture) event can be easily identified from the displacement-force data as shown in the figure. Based on the data obtained, the fracture point of the pillar microneedles was found to be 100 mN on average. Since only about 40 mN of force is required to penetrate the stratum corneum, the microneedles are strong enough to penetrate human skin without fracture. Furthermore, the similarity between the microneedle tips and the acrylic artifact cannot be fully established, so the actual fracture limit is likely much higher than 100 mN (i.e., the microneedles broke in a sequential manner rather than simultaneous fracture of most / all microneedles).

[0139] The pyramidal microneedle exhibited a continuously increasing force signature without any obvious sign of a breaking point.To identify the breaking limit of the pyramidal microneedle, an interrupted test was performed in which the microneedle was advanced and retracted into the artifact by a certain amount and examined by optical microscope images.This process was continued until breaking was observed.For this purpose, breaking was defined as bending of the pyramidal microneedle more than 15 degrees.

[0140] To further analyze the fracture of the microneedles, a finite element model (FEM) of the microneedle array shown in Figure 23 was developed. A series of nanoindentation tests (using a Hysitron nanoindenter) were performed to obtain the mechanical properties (elastic modulus and strength limit) of the CMC material. The average elastic modulus and yield strength of the CMC material (as prepared) were 10.8 GPa and 173 MPa, respectively. This indicates that the prepared CMC material has a higher elastic modulus and yield strength than both PMMA (elastic modulus: 3.1 GPa, yield strength: 103 MPa) and polycarbonate (elastic modulus: 2.2 GPa, yield strength: 75 MPa), indicating the superior strength and stiffness of the CMC material against other polymers.

[0141] Using this data, a series of FEM simulations were performed. The fracture limits of pyramidal and sharp pillar (width = 134 μm) microneedles with height 600 μm, apex angle 30 degrees, and fillet radius 20 μm were predicted from the FEM model to be 400 mN (pyramid) and 290 mN (sharp pillar) for asymmetric loading (5 degree loading misorientation). Considering that the minimum drilling force requirement is about 40 mN, pyramidal and sharp pillar microneedles should have a factor of safety of about 10 and 7.25, respectively.

[0142] When the fillet radius was doubled to 40 μm, the failure load of the pillar increased to 350 mN, and when the fillet radius was reduced to 5 μm, the failure load reduced to 160 mN, which is close to the experimentally determined failure load. The height and width of the pillar had a significant effect on the failure load. For example, for a 100 μm wide pillar, increasing the height from 500 μm to 1000 μm reduced the failure load from 230 mN to 150 mN. When the width was reduced to 75 μm for a 750 μm tall pillar, the failure load was found to be 87 mN.

[0143] To evaluate penetration capabilities, pyramid and sharp pillar microneedle arrays were tested for perforation against water-based model elastic substrates and full thickness human skin. Figure 24 shows stereomicrographs of pyramid (panels A, C, and E) and pillar microneedle arrays (B, D, and F) after 4 minutes of exposure to model elastic. In particular, toluene blue tracer dye was deposited on model elastic substrates (panels C and D) or freshly excised full thickness human skin explants (panels E and F) after application of pyramid or pillar microneedle arrays.

[0144] The model elastic substrate contained about 10% CMC and about 10% porcine gelatin in PBS that gelled for about 24 hours or more at about 4 degrees Celsius. The surface of the elastic was covered with about 100 μm thick parafilm to prevent immediate contact of the needle tip and the patch material with the water-based model elastic. Trypan blue tracer dye (Sigma Chem., Cat. No. T6146) was incorporated into the CMC hydrogel at a concentration of 0.1% to allow for stereomicroscopic imaging. Patches were applied using a spring-loaded applicator and analyzed after about 4 minutes of exposure. Based on the physical findings of the dye in the target substrate, the dissolution of the two different geometries of microneedles was significantly different.

[0145] Sharp post needles applied to the model elastic substrate released substantially more tracer dye into the gel matrix than was observed with the pyramidal design (Figure 24, C vs. D). Images of retrieved patches (Figure 24, A vs. B) are consistent with this finding, since degradation of the sharp post needles outpaced degradation of the pyramidal needles. To extrapolate this analysis to a more clinically relevant model, pyramidal and post microneedle arrays were applied to freshly excised full-thickness human skin explants using the same force from a spring-loaded applicator. Consistent with the results from the elastic model, pyramidal microneedle arrays deposited visibly less tracer dye than sharp post microneedle arrays (Figure 24, E vs. F).

[0146] To further evaluate penetration and delivery efficacy to human skin, CMC microneedle arrays were fabricated using BioMag (Polysciences, Inc., Catalog No. 84100) beads or fluorescent particulate tracers (Fluoresbrite YG 1 μm, Polysciences Inc., Catalog No. 15702). Pyramidal CMC microneedle arrays containing fluorescent or solid particulates were applied to living human skin explants as previously described. Five minutes after application, surface residues were removed and skin samples were cryosectioned and then counterstained with toluene blue for imaging by light microscopy (FIG. 25A and FIG. 25B) or fluorescence microscopy (FIG. 25C).

[0147] Pyramidal CMC microneedles effectively penetrated the stratum corneum, epidermis and dermis of living human skin explants, as evidenced by the deposition of BioMag beads lining the penetration cavities corresponding to the individual needle insertion points (representative sections shown in Figures 25A and 25B). Notably, well-ordered cavities (Figure 25A, cavities numbered 1-4, toluene blue counterstain, 10x) and the deposition of BioMag particles (brown) lining the penetration cavities were evident (Figure 25B, 40x), indicating that the microneedles penetrated human skin. Furthermore, analysis of sections from living human explants stained with DAPI to identify cell nuclei and anti-HLA-DR to identify MHC class II+ antigen-presenting cells revealed dense fluorescent granules deposited in the superficial epidermis and dermis, including some particles that colocalized with class II+ antigen-presenting cells (Figure 25C, DAPI (blue), HLA-DR+ (red), and fluorescent particles (green), 40x).

[0148] These results further demonstrate that the CMC microneedle arrays described herein can effectively penetrate human skin and deliver integrated cargo (bioactive components), including insoluble particulates, consistent with the effective delivery of particulate antigens to antigen-presenting cells in human skin, a current major goal of rational vaccine design.

[0149] To further address microneedle array delivery in vivo, dermal delivery of particulate antigens in vivo was modeled by similarly applying fluorescent particle-containing arrays to the dorsal aspect of the ear of anesthetized mice. After 5 minutes, the patch was removed and the mouse resumed its normal activities. At 3 hours or 3 days, ear skin and draining lymph nodes were analyzed for the presence of fluorescent particles. Consistent with findings in human skin, particulates were evident in skin excised from the array application site (data not shown). Furthermore, at the 3 day time point, substantial numbers of particles were evident within the draining lymph node. Figures 26A and 26B show substantial numbers of particles that were evident within the draining lymph node (Figure 26A, 10x), including clusters of particulates closely associated with class II+ cells (Figure 26B, 60x), suggesting the presence of lymph node-resident antigen-presenting cells that had internalized the particulates.

[0150] To quantitatively evaluate the effect of needle geometry on cargo delivery using microneedle arrays, 3 H tracer-labeled CMC microneedle arrays were constructed. CMC hydrogels were prepared with 5% wt of ovalbumin as the model active ingredient at a final dry weight content of 25 wt% (5 g / 95 g of OVA / CMC), 0.1 wt% trypan blue, and 3 0.5 × 10 in the form of H-thymidine (ICN Inc., catalog number 2406005) 6 dpm / mg dry weight 3 From a single batch of labeled CMC hydrogel preparation, four batches were prepared containing several individual patches of pyramid and sharp pillar needle geometries. 3 H-CMC microneedle arrays were fabricated. Patches were applied to human skin explants as described above and removed after 30 minutes of exposure. The patch-treated areas were tape-stripped to remove surface debris and cut using a 10 mm biopsy punch. 3 H content was determined by scintillation counting. 3The specific activity of the H-CMC microneedle patch material was determined and calculated to be 72,372 cpm / mg dry weight. This specific activity was used to indirectly determine the amount of ovalbumin delivered to and retained by the skin. The data obtained is summarized in Table 1 below.

[0151] The patch types tested were consistent between microneedle arrays (mean standard deviation 24-35%) and between batches (mean standard deviation 7-19%). The within-batch variability for both needle geometries was less than the in-batch values, indicating that the insertion process and target characteristics likely play a major role in successful transdermal material delivery and retention. The patch material retention data clearly demonstrates the primary importance of microneedle geometry in transdermal cargo delivery. Cylindrical needle geometries were overall 3.89 times larger than those of pyramidal needles. 3 Based on the deposited radioactive material, it is estimated that the pyramidal needles were inserted to a depth of approximately 200 μm, while the pillar-shaped needles were inserted approximately 400 μm or more.

[0152] Table 4.2.5. Pyramid and columnar needles for human skin explants 3 Transfer of H-labeled CMC microneedle material [Table 4]

[0153] Desirably, the microneedle arrays described herein can be used for cutaneous immunization. The development of strategies for effective delivery of antigens and adjuvants is a major goal in vaccine design, and immunization strategies that target cutaneous dendritic cells have various advantages over traditional vaccines.

[0154] Cancer treatment application The bioactive components used with the microneedle arrays described herein may include one or more chemotherapeutic agents. Effective and specific delivery of chemotherapeutic agents to tumors, including skin tumors, is a major goal of modern tumor therapy. However, systemic delivery of chemotherapeutic agents is limited by several well-established toxicities. In the case of skin tumors, including skin-derived tumors (such as basal cell, squamous cell, Merkel cell, and melanoma), as well as tumors metastatic to the skin (such as breast cancer, melanoma), local delivery may be effective. Current methods of local delivery generally require repeated application of creams or local injections. The effectiveness of these approaches is currently limited by limited penetration of active agents into the skin, non-specificity, and unwanted side effects.

[0155] The microneedle array of the present disclosure can be used as an alternative to or in addition to traditional local chemotherapy techniques.The microneedle array of the present disclosure can penetrate the outer layer of the skin and effectively deliver active biologics to living cells in the dermis and epidermis.The delivery of the chemotherapy agent leads to apoptosis and death of skin cells.

[0156] Furthermore, multiple bioactive agents can be delivered in a single microneedle array (patch). This allows for immunochemotherapeutic approaches based on the co-delivery of cytotoxic agents with immune stimulants (adjuvants). In the immunogenic environment created by the adjuvants, tumor antigens released from dying tumor cells are presented to the immune system, inducing local and systemic antitumor immune responses capable of rejecting tumor cells at the site of treatment and throughout the body.

[0157] Example 6 In an exemplary embodiment, the delivery of biologically active small molecules was tested. In particular, the activity of the chemotherapeutic agent Cytoxan® delivered to the skin with a CMC microneedle array was tested. The use of Cytoxan® allows for direct measurement of biological activity (Cytoxan®-induced apoptosis in the skin), representing a class of agents with potential clinical utility in the topical treatment of a range of cutaneous malignancies.

[0158] To directly evaluate the immunogenicity of CMC microneedle array-incorporated antigens, the well-characterized model antigen ovalbumin was used. Pyramid arrays were fabricated incorporating soluble ovalbumin (sOVA), particulate ovalbumin (pOVA), or arrays containing both pOVA along with CpG. The adjuvant effect of CpG has been well characterized in animal models, and their adjuvant activity in humans is currently being evaluated in clinical trials.

[0159] Immunization was achieved by applying antigen-containing CMC microneedle arrays to the ears of anesthetized mice using a spring-loaded applicator as described above, followed by removal of the array 5 minutes after application. These pyramidal microneedle arrays contained approximately 5 wt% OVA in CMC, and approximately 0.075 wt% (20 μM) CpG. As a positive control, a gene gun-based genetic immunization strategy using plasmid DNA encoding OVA was used. Gene gun immunization is one of the most potent and reproducible methods for inducing CTL-mediated immune responses in mouse models, suggesting its use as the "gold standard" for comparison in these assays.

[0160] Mice were immunized and boosted one week later, and then assayed for OVA-specific CTL activity in vivo. Notably, immunization with arrays containing small amounts of OVA and CpG induced high levels of CTL activity, similar to that observed by gene gun immunization (Figure 27). Significant OVA-specific CTL activity was elicited by both particulate and soluble array-delivered OVA antigen, even in the absence of adjuvant. It is well established that substantially higher doses of antigen are required for similar responses when delivered by traditional needle injection.

[0161] To evaluate the stability of the fabricated arrays, batches of arrays were fabricated, stored, and then used over time. As shown in Figure 28, no significant deterioration in immunogenicity was observed over the storage period up to 80 days (the longest time point evaluated). Therefore, the CMC microneedle array and this delivery technology can enable effective dermal delivery of antigens and adjuvants to induce antigen-specific immunity.

[0162] To evaluate the delivery of biologically active small molecules, pyramidal CMC microneedle arrays were fabricated containing the low molecular weight chemotherapeutic agent Cytoxan® (cyclophosphamide) or FluoresBrite green fluorescent particles as a control. Cytoxan® was integrated at a concentration of 5 mg / g CMC, allowing the delivery of approximately 140 μg per array. This is a therapeutically relevant concentration based on the area of ​​skin targeted, but well below the levels associated with systemic toxicity. Organ cultures of live human skin were used to evaluate the cytotoxicity of Cytoxan®. Cytoxan® was delivered by applying the arrays to skin explants as previously described by the inventors. After 5 minutes of application and 72 hours of exposure, the arrays and residual material were removed, and the cultured live skin explants were cryosectioned and fixed. Apoptosis was assessed using a green fluorescent TUNEL assay (In Situ Cell Death Detection Kit, TMR Green, Roche, Catalog Number: 11-684-795-910). As shown in Figure 29A, fluorescent microscopic image analysis of human skin sections revealed widespread apoptosis of epidermal cells in Cytoxan®-treated skin. As shown in Figure 29B, no visible apoptosis was observed in the skin treated with fluorescent particles, but the particles were evident, validating that the observed areas were accurately targeted by the microneedle array. Example 7

[0163] In another embodiment, localized treatment of established tumors with doxorubicin and / or poly(I:C) incorporated into MNAs established tumor regression and durable immunity capable of protecting against subsequent lethal systemic tumor challenge.

[0164] Novel therapeutic approaches for treating established skin tumors have been brought about based on the combined effects of MNA delivering chemotherapy, MNA delivering immunostimulant therapy, and / or combined chemo-immunotherapy. The B16 melanoma model was used as a model tumor to test these novel approaches. The B16 melanoma model is very well studied and is one of the most aggressive mouse skin cancers. Of all available skin tumor models, established B16 tumors are among the most difficult to treat. Moreover, B16 has a very high metastatic potential, allowing for a clinically relevant evaluation of systemic tumor immunity.

[0165] B16 skin tumors were established in normal mice by injection. Visible established skin tumors were treated once weekly for three weeks with MNAs containing doxorubicin alone, poly(I:C) alone, or doxorubicin and poly(I:C) incorporated in the same MNA. The doxorubicin dose selected corresponds to the MNA dose that induces apoptosis in human skin without causing necrosis. Tumor growth and survival were measured periodically for the duration of the study. As shown in Figures 41A and 41D, treatment with MNAs containing doxorubicin alone slowed tumor growth and improved survival (30%) compared to that observed in untreated tumor-bearing animals, where mortality was 100%. Furthermore, treatment with MNAs containing poly(I:C) alone slowed tumor growth (Figure 41B) and improved survival (50%) (Figure 41D) compared to that observed in untreated tumor-bearing animals, where mortality was 100%. Notably, treatment with both doxorubicin and poly(I:C) significantly slowed tumor growth in all animals (FIG. 41C) and completely eradicated tumors in 8 of 10 mice, reflected in an 80% long-term survival out to day 70 (FIG. 41D).

[0166] Representative images of control and treated animals are shown in Figures 42 and 43. Surviving animals were evaluated to determine whether they developed long-term immunity to these same tumors. Specifically, systemic immunity, including the durability of immune responses and the ability of surviving animals to survive IV challenge, was evaluated in these animals. Specifically, 60 days after the first MNA treatment, mice were treated with a lethal dose of B16. 14 days later, mice were sacrificed and lung metastases were quantified microscopically. Treated mice demonstrated a dramatic reduction in the number of lung lesions compared to naive controls (Figures 44-46). Taken together, these results demonstrate the ability of MNAs for delivering chemotherapeutic agents, immunostimulants, and combinations of these agents to both induce regression of established skin tumors and simultaneously induce durable, systemic, tumor-specific immune responses that can protect subjects from subsequent tumors.

[0167] In another embodiment, poly-ICLC can be substituted for poly(I:C), for example, forming an MNA comprising poly-ICLC in combination with at least one other chemotherapeutic agent (e.g., doxorubicin).

[0168] As discussed above, the one or more chemotherapeutic agents may include one or more immunostimulants (specific and non-specific) known to those skilled in the art to stimulate the immune system to reject and destroy tumors, such as poly(I:C) and poly-ICLC. These immunostimulants may be integrated into the MNA along with other chemotherapeutic agents, such as cytotoxic agents like doxorubicin. Immunostimulants that may be used in the manner described herein include adjuvants, Toll-like receptors (TLRs), ribonucleotides and deoxyribonucleotides, double-stranded RNA (dsRNA), and derivatives of poly(I:C).

[0169] The one or more chemotherapeutic agents may include various anthracycline agents. For example, as discussed above, doxorubicin may be integrated into the MNA. Other examples of anthracycline agents include, for example, daunorubicin, epirubicin, idarubicin, valrubicin, and mitoxantrone. In some embodiments, at least two different anthracycline agents may be integrated into the same MNA. As discussed in more detail herein, the MNA may be formed using CMC or other suitable polymers. Thus, at least one anthracycline agent (e.g., doxorubicin), or two or more different anthracycline agents may be integrated into the same MNA. More than one anthracycline drug (eg, doxorubicin and another anthracycline drug) can be incorporated into an MNA that includes a polymer such as CMC.

[0170] Directly fabricated microneedle arrays The micromilling of the mastermold described above allows for the production of microneedle arrays with various geometries. In another embodiment, a system and method are provided for fabricating microneedle arrays by directly micromilling various materials, such as dry CMC sheets. The same general tooling described above for micromilling the mastermold can be used to directly micromill the microneedle arrays.

[0171] Direct micromilling of microneedle arrays eliminates the need for a molding step and allows for a simplified, scalable, and precisely reproducible production strategy that is compatible with large-scale clinical applications. Furthermore, direct fabrication of microneedle arrays by micromilling allows for greater control of microneedle geometry. For example, micromilling allows for the inclusion of microneedles that retain features such as undercuts and / or bevels that cannot be achieved using molding processes.

[0172] The repeatability of direct milling of microneedle arrays is particularly beneficial; that is, in direct micromilling, all of the microneedles are identical as a result of the milling fabrication process. In molding operations, it is not uncommon for some needles to be missing or broken from a given patch as a result of the process of physically separating them from the mold. For use in certain medical applications, repeatability of the amount of bioactive component in the array is very important to provide an appropriate level of "quality control" over the process, since needle irregularities between patches would likely result in variability in the dose of drug / vaccine delivered. Of course, repeatability would also be an important advantage for any application requiring FDA approval. Spin-cast / molded patches would require special processes to ensure acceptable uniformity for consistent drug delivery. This quality control would also likely result in a certain percentage of patches "failing" this release test, introducing waste into the production process. Direct micromilling eliminates, or at least significantly reduces, these potential issues.

[0173] The molding process also has inherent limitations due to the need to fill wells or concavities and be able to remove the hardened molded part from the wells or concavities. That is, due to the geometry of the mold, undercuts must generally be avoided when molding the part or the part will not be removable from the mold. That is, a geometric limitation of a molded part, such as a molded microneedle array, is that any features located closer to the apex must be narrower than any features located toward the base.

[0174] Thus, with these limitations in mind, Figure 20 shows a schematic representation of a microneedle shape and structure that is generally suitable for fabrication by molding. That is, the shapes and structures shown in Figure 20 do not contain any undercuts that would prevent the part (i.e., the microneedle) from being removed from the production mold. In contrast, Figure 30 shows an angled undercut microneedle shape that cannot be molded in the manner described herein.

[0175] This geometry can only be made by direct fabrication using the proposed micromilling technique. The negative (bevel) angle promotes better retention of the microneedle in the tissue. Furthermore, since the microneedle of FIG. 30 has a wider middle section (with a larger cross-sectional dimension) above the lower section (with a smaller cross-sectional dimension), a larger amount of bioactive material can be delivered by configuring the microneedle to hold or store the bioactive material in the wider section that is configured to be retained in the skin. Thus, the larger cross-sectional dimension of the middle section can "carry" the bulk of the bioactive ingredient. Since the lower section tapers to a narrower cross-sectional dimension, the wider middle section will have better penetration to deliver the bioactive ingredient into the skin layer. The portion above the middle section is preferably tapered to promote the penetration of the microneedle into the skin layer.

[0176] Another limitation of molded parts is that it may be difficult to fill very small sections of the mold accurately. Since a production mold for a microneedle array contains many very small sections, it may be difficult to fill each well accurately. This may be particularly problematic when the mold must be filled with different materials, such as a material that contains a bioactive component and a material that does not contain a bioactive component. Thus, when the production mold is filled in layers, it may be difficult to accurately fill the very small wells associated with each microneedle. Such reproducibility is particularly important because the microneedles are intended to deliver one or more bioactive components. Thus, even a small variation in the amount of bioactive component used to fill the production mold may be highly undesirable.

[0177] Also, by using a laminated structure to form a sheet or block that can be micromilled, various active ingredients can be integrated into a single microneedle by vertical layering. For example, in an exemplary embodiment, CMC hydrogel and CMC-sOVA hydrogel (80% CMC / 20 wt% OVA) are layered into a sheet or block form. This composite sheet can be micromachined using the direct micromilling technique described herein.

[0178] Figure 31 is a stereo microscope image analysis of an entire microneedle array. The microneedles comprise a 10 x 10 array of microneedles. Figure 32 is a magnified segment of the microneedle array of Figure 31. The layering of the two components is shown in Figure 32, which shows the darker areas of the microneedles at the tip and the lighter areas of the microneedles at the base. The darker layer at the tip represents the layer containing the bioactive component, in this case soluble ovalbumin contained in a CMC layer.

[0179] Although the formation of a layer containing an active material (e.g., an antigen) and subsequent micromilling of this layer (and any other adjacent layers) may require the use of relatively large amounts of active material, the material can be removed (e.g., in the form of chips), salvaged, and reused. Direct mechanical cutting techniques are not limited by the geometric constraints that arise from molding / demolding approaches, and therefore can create more innovative needle designs (e.g., FIG. 30), which can significantly improve the retained needle volume and needle retention time within the skin.

[0180] Producing a sheet or block by forming multiple layers can result in a solid material that can be micromachined and can include one or more layers with bioactive components. For example, a soluble solid carboxymethylcellulose polymer-based block or sheet with well-defined and controlled dimensions can be fabricated by a lamination process. The resulting sheet or block is fully machineable, similar to the machining of a plastic or metal sheet or block. As described herein, this fabrication process may be suitable for incorporating bioactive components into the matrix without significantly reducing their activity level.

[0181] As described below, the fabricated sheets of material (such as CMC-based materials) can be directly micromachined / micromilled to produce one or more microneedle arrays suitable for delivering active components through the skin. This dissolvable biocompatible CMC block material can be used to deliver soluble or insoluble and particulate agents in a sustained release manner for application to body surfaces.

[0182] Biocompatible materials may be suitable for implantation into deeper soft or hard tissues where dissolution of the scaffolding material is required and useful.

[0183] The following method can be used to prepare carboxymethylcellulose (CMC) polymer low viscosity hydrogel to a concentration of 12.5%. 12.5% ​​carboxymethylcellulose (CMC) low viscosity hydrogel can be prepared in water or other biocompatible buffers such as (but not limited to) PBS or HBS. During the preparation of the polymer solution, soluble agents (such as nucleic acids, peptides, proteins, lipids, or other organic and inorganic biologically active components) and particulates can be added (e.g., ovalbumin, soluble agents). Ferrous particulates can be used that carry active components at 20 w / w% of CMC.

[0184] The preparation of 1000 g of sterile 12.5% ​​CMC hydrogel without any active ingredient can be achieved as follows: 1) Measure 125g of CMC and add 875g of water or other water-based solvent. 2) Blend with an overhead mixer until homogenous. 3) Sterilize the homogenate by autoclaving at 121 degrees Celsius for 1 hour (the autoclaving step can reduce viscosity and improve stratification). 4) Cool to 22 degrees Celsius. 5) The resulting material is vacuum treated at 10 Torr and 22 degrees Celsius for 1 hour to remove trapped microbubbles. 6) Centrifuge the product at 25,000g for 1 hour in a centrifuge with a vacuum chamber (to float and further remove any remaining microbubbles). 7) Store the CMC hydrogel product at 4 degrees Celsius.

[0185] Preparation of 1000 g of sterile 20 / 80% ovalbumin / CMC hydrogel with a dry content of 12.5 w / w% can be achieved as follows: 1) Measure out 100g of CMC and add 650g of water or other water-based solvent. 2) Blend with an overhead mixer until homogenous. 3) Sterilize the homogenate by autoclaving at 121 degrees Celsius for 1 hour (this autoclaving step can reduce viscosity and improve stratification). 4) Cool to 22 degrees Celsius. 5a) Dissolve 25 g of ovalbumin in 225 g of water. 5b) Sterile filter the ovalbumin solution through a 0.22 μm pore size filter. 6) 750 g of CMC hydrogel is mixed with 250 g of sterile ovalbumin solution under sterile conditions until homogenous. 7) The resulting material is vacuum treated at 10 Torr and 22 degrees Celsius for 1 hour to remove trapped microbubbles. 8) Centrifuge the product at 25,000g for 1 hour in a centrifuge with a vacuum chamber (to float and further remove any remaining microbubbles). 9) Store the CMC hydrogel product at 4 degrees Celsius.

[0186] Preparation of 100 g of sterile 20 / 80% granular ovalbumin / CMC hydrogel with a dry content of 12.5 w / w% can be achieved as follows: 1) Measure out 10 g of CMC and add 87.5 g of water or other water-based solvent. 2) Blend with an overhead mixer until homogenous. 3) Sterilize the homogenate by autoclaving at 121 degrees Celsius for 1 hour (this autoclaving step can reduce viscosity and improve stratification). 4) Cool to 22 degrees Celsius. 5) Disperse 2.5 g of granular ovalbumin into 97.5 g of CMC hydrogel at 22 degrees Celsius and mix under sterile conditions until homogenous. 6) The resulting material is vacuum treated at 10 Torr and 22 degrees Celsius for 2 hours to remove trapped microbubbles. 7) Centrifuge the product at 3,000g for 1 hour in a centrifuge with a vacuum chamber (to float and further remove any remaining microbubbles). 8) Store the CMC hydrogel product at 4 degrees Celsius.

[0187] It should be noted that in this example, particulate ovalbumin is prepared from the reaction of activated iron beads to ovalbumin, however, it should be noted that the above description is only an exemplary embodiment and other compounds and active ingredients may also be used.

[0188] A solid block / sheet of carboxymethylcellulose (CMC) can be fabricated using the low viscosity CMC hydrogel described above in the following manner.

[0189] The fabrication process may include spreading a layer of polymer at a defined thickness and drying the layered polymer to a moisture content of less than about 5% using a stream of sterile dry air on the surface of the polymer layer. The above two actions may be repeated until the desired block thickness is achieved.

[0190] A method for achieving a laminar CMC hydrogel layering of a defined thickness in a mold assembly is described with reference to Figure 33. Figure 33 shows a cross-sectional view of a mold assembly including (a) a casting bed; (b) an adjustable casting bed wall; (c) a casting bed depth adjustment assembly; and (d) an acrylic spreader. It should be noted that Figure 33 is not drawn to scale or otherwise shows elements in proper proportion.

[0191] The mold assembly may be constructed from acrylic (plexiglass) and may include a casting bed base unit, a vertically adjustable hydrophobic casting bed wall, and a casting bed adjustment mechanism. The casting bed base unit (a1) may include a removable / replaceable casting bed top plate (a2) to which a cellulose layer (a3) ​​is attached. The cellulose layer may be approximately 0.5 mm thick. The vertically adjustable hydrophobic casting bed wall (b) may be adjusted using a casting bed depth adjustment mechanism that may consist of a lead screw (c1) and a level adjustment knob (c2). In the embodiment shown, a quarter turn of this knob may raise the bed wall by 0.5 mm.

[0192] Initially, the adjustable casting bed walls can be set to a height where the distance between the acrylic spreader and the cellulose layer of the bed is about 1 mm when the spreader is in place. A predefined volume (e.g., about 0.1 ml / cm) can be added. 2 ) of 12.5% ​​CMC hydrogel can be added and layered. The layer can be smoothed or flattened by sliding an acrylic spreader (d) on the top surface of the adjustable casting wall to obtain a smooth layer of CMC hydrogel of about 1 mm. The layered CMC hydrogel can be dried to a solid phase in a drying apparatus shown in FIG. 34 and described in more detail below.

[0193] The layering and drying steps can be repeated until the desired layered structure (sheet) is achieved. The casting bed walls can be raised by an appropriate amount between the addition of each layer. For example, after each layer is added, the bed walls can be raised or lifted by about 0.5 mm. Thus, the cycle described above can deposit a solid CMC layer of about 0.5 mm. This process (e.g., layering of material, raising of bed walls, etc.) can be repeated until the desired block thickness is achieved.

[0194] The layered CMC hydrogel polymer can be dried in a variety of ways. For example, FIG. 34 shows a drying apparatus that can be used to dry various stacked layers of sheet material. It should be noted that FIG. 34 is not drawn to scale or otherwise shown with elements in proper proportion. A fan can provide a continuous gas flow (e.g., air or other inert gas such as nitrogen) over the layered CMC hydrogel in the mold assembly. The gas flow gently dehydrates the CMC hydrogel layers. The drying rate can be adjusted to prevent or reduce gas inclusions (e.g., air bubbles) in the solid CMC product. The high humidity air over the layers can be dried with a desiccant (e.g., air dryer or dehumidifier), temperature adjusted, and returned again over the hydrogel by a speed-controlled fan. A hygrometer can be placed in place on the high humidity side of the chamber to indicate the status of the drying process. After the desired dryness is achieved, as indicated by the hygrometer, the drying process can be terminated.

[0195] Airflow can be adjusted to affect the drying rate. In an exemplary embodiment, airflow is controlled between about 0.1-2.0 m / sec and the temperature is between ambient and about 50 degrees Celsius. Using these configurations, drying times for single layer CMC hydrogels can be about 0.5-4 hours depending on airflow and set temperature.

[0196] The pure CMC-based product may be clear, pale off-white, or amber in color. Its specific gravity may be about 1.55-1.58 g / ml. The product should be free of microbubbles and otherwise suitable for fabricating micron-scale objects. The physical characterization (hardness, tensile strength, etc.) of the final block / sheet product may vary, but generally should be able to withstand the physical stresses associated with micromilling.

[0197] As described above, the microneedle arrays disclosed herein can provide a reliable and precise delivery method for various bioactive ingredients. The structural, manufacturing and distribution advantages characteristic of the microneedle arrays described above may be particularly applicable for use in vaccine delivery. The advantages of these microneedle arrays include (1) safety, which eliminates the need for needles or live vectors for vaccine delivery; (2) economy, due to inexpensive production, product stability and ease of distribution; and (3) versatility, via a delivery platform that is compatible with a variety of antigen and adjuvant formulations.

[0198] Moreover, immunization of the skin with microneedle arrays has important advantages in immunogenicity. Skin is rich in easily accessible dendritic cells (DCs) and has long been regarded as a highly immunogenic target for vaccine delivery. These dendritic cell populations constitute the most potent antigen-presenting cells (APCs) identified to date. For example, genetic immunization of the skin transfects and activates mouse and human dermal dendritic cells, which synthesize transgenic antigens, migrate to skin-draining lymph nodes, and efficiently present these antigens by an MHC class I-restricted pathway to stimulate CD8+ T cells. The immune responses induced by skin-derived DCs are remarkably potent and long-lasting compared to those induced by other immunization techniques. Recent clinical trials have demonstrated that even conventional vaccines are significantly more potent when delivered intradermally rather than by standard intramuscular needle injection. Thus, microneedle arrays can efficiently and simultaneously deliver both antigens and adjuvants, allowing both targeting of DCs and adjuvant engineering of the immune response using the same delivery platform.

[0199] High Frequency Electromagnetic Vibration Applicator The microneedle array device can be applied to human skin by various methods, including self-application or assisted application by human pressure (e.g., pressing with a finger or thumb) or with a spring-loaded device. To facilitate the ease and reproducibility of delivery of the microneedle array device, including tip-loaded microneedle arrays, an applicator device is described herein. The applicator device is configured to convert high-frequency electromagnetic vibrations into unidirectional mechanical resonance of the active head. This in turn allows multiple reproducible low-amplitude and high-frequency pressure strokes, which facilitate the insertion of the microneedles of the microneedle array into tissue, including human skin.

[0200] As shown in Figure 38, the applicator can include an applicator head, an oscillator-energy converter, an electromagnetic oscillator, and a power source. If desired, one or all of these four elements can be separable from the applicator device.

[0201] The applicator heads can be interchangeable to accommodate and act on tissue surface areas of different sizes and shapes. As shown in FIG. 39, a variety of applicator head geometries can be utilized in combination with the applicators described herein. The applicator heads are interchangeably made of stainless steel or other chemically and physically resistant materials. If necessary, the applicator heads can be autoclaved for sterility and / or sterilized with alcohol or other chemicals. Alternatively or additionally, gas sterilization (ethylene oxide) is possible.

[0202] Application specific geometries can be rapidly designed and fabricated. For example, the area of ​​a single application in this example is 5 mm2 depending on the geometry of the active head. 2 ~250mm 2 A wide range can be achieved by simple structural variations in the head geometry.

[0203] The oscillator energy converter unit can be configured to convert the electromagnetic vibrations into a mechanical movement of the applicator head. The amplitude of the applicator head in direction Z can be controlled between 0.5 and 2.5 mm (Figure 40; A). In some embodiments, the movement of the head in direction XY can be configured to be negligible, i.e. less than 0.2 mm (Figure 40; B). The frequency of the mechanical movement resulting from the energy conversion in direction Z can be controlled between 500 and 25000 rpm. Optionally, the oscillator energy converter unit can be separable and can be disposed of or sterilized if necessary.

[0204] An electromagnetic (EM) oscillator may be made up of three subunits. These subunits may include: (1) a controller and a regulated power supply that generates the voltage and power for the high frequency EM oscillator, (2) a controller-regulator that generates the high frequency signal and the required current for the EM oscillator, and (3) the EM oscillator. The output frequency can be controlled by the user (e.g., within a range such as 100-500 Hz). In some embodiments, the EM oscillator may be completely sealed and can be sterilized by alcohol solution or other chemical agents.

[0205] The power supply unit also supports different power sources, e.g.: A. Regular disposable alkaline or any other type of battery; b. Rechargeable NiCad or Li-oxide batteries with built-in inductive charger; c. 100~240V electronic power adapter etc., may be separable to accommodate.

[0206] The applicator can provide several benefits in conjunction with microneedle array application. For example, the applicator can minimize the mechanical force required for microneedle array insertion into tissue. The applicator can also reduce the effects of pain compared to existing spring-loaded applicators. Additionally, the applicator can be portable, and the components of the applicator can be separable and interchangeable. Finally, the applicator can be configured such that it can be sterilized for aseptic use.

[0207] Considering the many possible embodiments to which the principles of the disclosed embodiments may be applied, it should be appreciated that the illustrated embodiments are merely preferred examples and should not be interpreted as limiting the scope of protection. Rather, the scope of protection is defined by the following claims. Accordingly, the inventors claim all that comes within the scope and spirit of these claims.

Claims

1. 1. A microneedle array for treating skin cancer in a target skin area, comprising: a microneedle array matrix having a cargo incorporated therein, said cargo comprising a dissolvable biocompatible material and a therapeutically effective amount of at least two chemotherapeutic agents, said at least two chemotherapeutic agents comprising a cytotoxic agent and an immunostimulatory agent; A base and a plurality of microneedles extending from the base; Including, the at least two chemotherapeutic agents are located within the plurality of microneedles, and the base is free of the at least two chemotherapeutic agents; a subject having skin cancer is selected, and the microneedle array is inserted into the target skin area of ​​the subject; the act of inserting a microneedle array includes penetrating the stratum corneum to deliver the at least two chemotherapeutic agents to the epidermis and / or dermis, providing localized dermal delivery of a therapeutically effective amount of the at least two chemotherapeutic agents to the subject without systemic exposure; the treatment induces regression of one or more established skin tumors in the target skin area caused by the skin cancer. Microneedle array.

2. The microneedle array of claim 1 , wherein the cytotoxic agent comprises a therapeutically effective amount of doxorubicin.

3. The microneedle array of claim 1 , wherein the at least two chemotherapeutic agents comprise doxorubicin and a second chemotherapeutic agent.

4. The microneedle array of claim 1 , wherein the dissolvable biocompatible material is carboxymethyl cellulose.

5. The microneedle array of claim 1 , wherein the at least two chemotherapeutic agents include doxorubicin and further include at least one other anthracycline agent.

6. The microneedle array of claim 1 , wherein the one or more immunostimulatory agents include at least one adjuvant.

7. The microneedle array of claim 1 , wherein the one or more immunostimulatory agents comprise at least one TLR agonist.

8. The microneedle array of claim 1 , wherein the one or more immunostimulatory agents comprise at least one of ribonucleotides or deoxyribonucleotides.

9. The microneedle array of claim 1 , wherein the one or more immunostimulatory agents comprise at least one dsRNA.

10. The microneedle array of claim 1 , wherein the one or more immunostimulatory agents comprise at least one poly(I:C) derivative.

11. The microneedle array of claim 10 , wherein the at least one poly(I:C) derivative comprises poly(I:C).

12. The microneedle array of claim 10, wherein the at least one poly(I:C) derivative comprises poly-ICLC.

13. The microneedle array of claim 10, wherein the at least one poly(I:C) derivative comprises poly(I:C) and poly-ICLC.

14. The microneedle array of claim 10, wherein the therapeutically effective amount of the at least one poly(I:C) derivative is about 250 micrograms.

15. The microneedle array of claim 1 , further comprising one or more viral vectors.

16. The microneedle array of claim 2 , wherein the therapeutically effective amount of doxorubicin is about 50 or 200 micrograms.

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