Silk fibroin-based microneedles and uses thereof

Abstract

Microneedles and microneedle devices comprising, for example, a silk fibroin-based microneedle tip for sustained transdermal delivery of an anti-cancer agent and / or an immunomodulatory agent, and methods of making and using the same are described herein. In other embodiments, compositions and methods for the sudden release or sustained release administration of an anti-cancer agent and / or an immunomodulatory agent to provide an improved immune response to cancer in a subject are described.

Description

[0001] This invention is a divisional application of the invention patent application filed on October 9, 2020, with application number 202080071018.4 and invention title "Microneedles based on silk fibroin and their uses". Related applications

[0002] This application claims priority to U.S. Serial No. 62 / 912,832, filed October 9, 2019. The contents of the foregoing application are incorporated herein by reference in their entirety. Technical Field

[0003] This disclosure generally relates to silk fibroin-based microneedles configured to release therapeutic agents, such as anticancer agents, immunomodulators, or combinations thereof, to treat subjects with diseases or conditions such as cancer. Background Technology

[0004] Due to the highly efficient barrier nature of the stratum corneum, the outermost layer of the skin, delivering drugs into the skin to produce local or systemic effects is extremely difficult. Microneedle devices, comprising submillimeter needles, are designed to be minimally invasive and bypass the stratum corneum to enter the skin's microcirculation for local and / or systemic delivery of therapeutic agents via the transdermal route. However, conventional microneedle designs and materials come with various limitations that hinder their production and limit their performance (see, for example, Donnelly et al., DrugDeliv. 17(4): 187-207, 2010). Specifically, it is difficult to design a microneedle that possesses both sufficient mechanical strength to pierce the stratum corneum and the ability to introduce and subsequently release an effective amount of therapeutic agent. Improved microneedle design and manufacturing methods are needed. Summary of the Invention

[0005] This disclosure is based, at least in part, on the understanding that silk fibroin possesses properties suitable for microneedle manufacturing, including all-aqueous processing, mechanical strength, biocompatibility, and the ability to stabilize and control the release of various therapeutic agents from a silk-based matrix. Additionally, this disclosure is based, at least in part, on the understanding that topical application of therapeutic agents such as anticancer agents and / or immunomodulators to tumor sites can lead to inhibition of tumor growth at or near the application site and can also induce a systemic immune response that ablates tumors at distant sites. Furthermore, certain diseases, such as cancer, are associated with tumor-specific antigens (e.g., neoantigens) that are poorly and / or inefficiently presented to the subject's immune system by antigen-presenting cells (APCs), for example, due to immunosuppression of the tumor microenvironment (TME). Not wishing to be bound by theory, this disclosure provides fibroin-based microneedles and fibroin-based microneedle devices that can be used, for example, to alter the tumor microenvironment by administering effective amounts of anticancer agents, immunomodulators, or combinations thereof, thereby promoting the presentation of tumor-specific antigens (e.g., neoantigens) to APCs in a subject and generating robust and durable immunity against tumor-specific antigens (e.g., cancer immunity). In some embodiments, fibroin-based microneedles and fibroin-based microneedle devices can be used to deliver patient-specific neoantigens (e.g., cancer vaccines) to achieve enhanced immunity via sustained release of therapeutic agents such as anticancer agents and / or immunomodulators in a subject.

[0006] This disclosure provides silk fibroin-based microneedles and silk fibroin-based microneedle devices configured to introduce and subsequently release (e.g., administer) an effective amount of a therapeutic agent or combination of therapeutic agents to a subject (e.g., a human subject). In some embodiments, the silk fibroin-based microneedles and silk fibroin-based microneedle devices comprise an anticancer agent, an immunomodulatory agent, or a combination thereof. In some embodiments, the microneedles disclosed herein may be used in combination with a second therapeutic agent or procedure such as cancer therapy (e.g., one or more of anticancer agents, immunotherapy, photodynamic therapy (PDT), surgery, and / or radiation).

[0007] The disclosed silk fibroin-based microneedles can be configured to release a therapeutic agent or combination of therapeutic agents (e.g., anticancer agents, immunomodulators, or combinations thereof) according to various release kinetics such as sudden release (e.g., immediate or rapid dissolution upon application to biological barriers such as skin, mucous surfaces, tumors, oral cavity, or buccal cavity, typically occurring within minutes) and / or sustained release. Examples of sustained release include, but are not limited to, zero-order release (e.g., the release rate is independent of the therapeutic agent concentration in the dosage form, e.g., the release rate is substantially constant over a period of time, e.g., a constant amount of therapeutic agent is eliminated per unit time), first-order release (e.g., the release rate is a function of the amount of therapeutic agent remaining in the dosage form, e.g., a constant percentage of the drug is eliminated per unit time), and second-order release (e.g., where doubling the therapeutic agent concentration in the dose would increase the release rate fourfold). In some embodiments, a portion of the microneedle is configured for a first type of release, such as sudden release, while another portion of the microneedle is configured for a second type of release, such as sustained release. Furthermore, the release (e.g., administration) of therapeutic agents (e.g., anticancer agents, immunomodulators, or combinations thereof) from the silk fibroin-based microneedles described herein can be facilitated by: diffusion of the therapeutic agent from the microneedles or a portion thereof; degradation of the microneedles or a portion thereof (e.g., protease-mediated degradation); and / or dissolution of the microneedles or a portion thereof.

[0008] The disclosed silk fibroin-based microneedles can be configured to possess sufficient mechanical properties (e.g., strength) and suitable geometry (e.g., tip sharpness, tip angle, length, needle spacing) to pierce biological barriers (e.g., skin, tumor, tissue, cell membrane, mucous surface, oral cavity, or buccal cavity) to achieve local and / or systemic delivery of therapeutic agents or combinations of therapeutic agents (e.g., anticancer agents, immunomodulators, or combinations thereof) to a subject. In some embodiments, the microneedles or devices described herein are configured to administer therapeutic agents (e.g., anticancer agents, immunomodulators, or combinations thereof) to tumor sites (e.g., within and / or around the tumor). In some embodiments, the microneedles or devices described herein are configured to administer therapeutic agents (e.g., anticancer agents, immunomodulators, or combinations thereof) to skin lesion sites.

[0009] In some embodiments, topical application of therapeutic agents (e.g., anticancer agents, immunomodulators, or combinations thereof) to the site of tumors and / or skin lesions via silk fibroin-based microneedles or silk fibroin-based microneedle devices described herein may induce anticancer effects (e.g., inhibition of tumor growth) at or near the application site, and optionally may induce systemic anticancer effects (e.g., immune responses) to ablate tumors at distant sites.

[0010] In some implementations, silk fibroin-based microneedles and silk fibroin-based microneedle devices are used in combination with standard care treatments (e.g., standard care treatments for cancer or skin conditions), which are optionally selected from surgery, chemotherapy, immunotherapy, targeted therapy, hormone therapy, and / or radiation therapy.

[0011] They also disclosed methods for manufacturing and using such microneedles to treat diseases such as cancer in subjects.

[0012] Those skilled in the art will recognize or be able to identify numerous equivalents of the specific embodiments of this disclosure described herein using experiments no more than those of conventional methods. Such equivalents are intended to be covered by the following embodiments (E).

[0013] E1. A microneedle device (e.g., a microneedle patch) comprising a plurality of silk fibroin-based microneedles, wherein the plurality of microneedles comprises: The first microneedle containing anticancer agents; and The second microneedle contains an immunomodulator. Optionally, a third microneedle may contain anticancer agents and / or immunomodulators. The first and / or second microneedles contain silk fibroin (e.g., regenerated silk fibroin and / or recombinant silk fibroin), and the microneedle device is configured to deliver the anticancer agent and the immunomodulator to the subject.

[0014] E2. A microneedle device (e.g., a microneedle patch) comprising a plurality of silk fibroin-based microneedles, wherein the plurality of microneedles comprises: Two or more microneedles containing anticancer agents, The two or more microneedles contain silk fibroin (e.g., regenerated silk fibroin and / or recombinant silk fibroin), and the microneedle device is configured to deliver the anticancer agent to the subject.

[0015] E3. A microneedle device (e.g., a microneedle patch) comprising a plurality of silk fibroin-based microneedles, wherein the plurality of microneedles comprises: Two or more microneedles containing immunomodulators, The two or more microneedles contain silk fibroin (e.g., regenerated silk fibroin and / or recombinant silk fibroin), and the microneedle device is configured to deliver the immunomodulator to the subject, optionally wherein the immunomodulator enhances the immune response against cancer.

[0016] E4. The microneedle device according to any one of the foregoing embodiments, wherein the first and / or second microneedle among the plurality of microneedles comprises: (i) Substrate (e.g., a soluble substrate), (ii) A silk fibroin tip (e.g., an implantable silk fibroin tip) comprising silk fibroin applied to a substrate, and (iii) (Optional) Applying a backing to the substrate.

[0017] E5. Multiple microneedles, wherein the multiple microneedles comprise: The first microneedle containing anticancer agents; and The second microneedle contains an immunomodulator. Optionally, a third microneedle may contain anticancer agents and / or immunomodulators. The first and / or second microneedles contain silk fibroin, such as regenerated silk fibroin and / or recombinant silk fibroin.

[0018] E6. Multiple microneedles, wherein the multiple microneedles comprise: The first microneedle containing anticancer agents; and The second microneedle contains an anticancer agent. The first and / or second microneedles contain silk fibroin, such as regenerated silk fibroin and / or recombinant silk fibroin.

[0019] E7. Multiple microneedles, wherein the multiple microneedles comprise: First microneedles containing immunomodulators; and The second microneedle contains an immunomodulator. The first and / or second microneedles contain silk fibroin, such as regenerated silk fibroin and / or recombinant silk fibroin.

[0020] E8. The microneedle device according to embodiment E4, wherein the silk fibroin tip contains an anticancer agent and / or an immunomodulator.

[0021] E9. The microneedle device according to embodiment E4, wherein the substrate comprises an anticancer agent and / or an immunomodulator.

[0022] E10. A microneedle device or a plurality of microneedles according to any one of the foregoing embodiments, wherein the microneedles are configured to pierce a biological barrier (e.g., skin).

[0023] E11. The microneedle device according to embodiment E4 or E8, wherein the microneedle is configured to implant a silk fibroin tip into the subject's biological barrier (e.g., skin).

[0024] E12. A microneedle device according to any one of embodiments E1-E4 or E8-E11, wherein the microneedle device is configured to deliver (e.g., release) an anticancer agent and / or immunomodulator to a subject locally and / or systemically.

[0025] E13. A microneedle device according to any one of embodiments E1-E4 or E8-E12, wherein the microneedle device is configured to deliver an effective amount of an anticancer agent and / or an immunomodulator to a subject.

[0026] E14. The microneedle device according to embodiment E4 or E8, wherein the silk fibroin tip comprises regenerated silk fibroin and / or recombinant silk fibroin.

[0027] E15. The microneedle device or multiple microneedles according to any one of the foregoing embodiments, configured to deliver (e.g., release) two or more anticancer agents (e.g., three or more, four or more, or five or more anticancer agents).

[0028] E16. The microneedle device or multiple microneedles according to any one of the foregoing embodiments, configured to deliver (e.g., release) two or more immunomodulators (e.g., three or more, four or more, or five or more immunomodulators).

[0029] E17. A microneedle device or a plurality of microneedles according to any one of the foregoing embodiments, wherein the plurality of microneedles includes at least one additional microneedle, wherein the additional microneedle contains the same first anticancer agent.

[0030] E18. A microneedle device or a plurality of microneedles according to any of the foregoing embodiments, wherein the plurality of microneedles includes at least one additional microneedle, wherein the additional microneedle contains an anticancer agent different from the first anticancer agent (“second anticancer agent”).

[0031] E19. A microneedle device or a plurality of microneedles according to any one of the foregoing embodiments, wherein the plurality of microneedles includes at least one additional microneedle, wherein the additional microneedle contains the same first immunomodulator.

[0032] E20. A microneedle device or a plurality of microneedles according to any of the foregoing embodiments, wherein the plurality of microneedles includes at least one additional microneedle, wherein the additional microneedle contains an immunomodulator different from the first immunomodulator (“second immunomodulator”).

[0033] E21. The microneedle device or plurality of microneedles according to any one of the foregoing embodiments, wherein the plurality of microneedles includes additional silk fibroin-based microneedles, the additional silk fibroin-based microneedles containing a second anticancer agent.

[0034] E22. The microneedle device or plurality of microneedles according to any one of the foregoing embodiments, wherein the plurality of microneedles includes additional silk fibroin-based microneedles, the additional silk fibroin-based microneedles including a second immunomodulator.

[0035] E23. The microneedle device or plurality of microneedles according to any one of the foregoing embodiments, comprising a plurality of the first, second and / or additional microneedles.

[0036] E24. A microneedle device or multiple microneedles according to any one of embodiments E1, E2, E4-E6 or E8-E23, wherein the anticancer agent is selected from one or more of small molecules (e.g., chemotherapeutic drugs), biological agents (e.g., antibodies), viral cancer therapeutics, nanomedicines and nucleic acid molecules (e.g., DNA and / or RNA).

[0037] E25. A microneedle device or multiple microneedles according to any one of embodiments E1, E2, E4-E6 or E8-E24, wherein the anticancer agent is not a chemotherapeutic nucleotide.

[0038] E26. A microneedle device or multiple microneedles according to any one of embodiments E1, E2, E4-E6 or E8-E25, wherein the anticancer agent is mRNA, optionally wherein the mRNA encodes an anticancer agent and / or an immunomodulator, optionally wherein the mRNA encodes a checkpoint inhibitor, TLR agonist, STING agonist, RIG agonist, cancer vaccine, targeted therapy and / or cytokine.

[0039] E27. A microneedle device or multiple microneedles according to any one of embodiments E1, E2, E4-E6 or E8-E26, wherein the anticancer agent is selected from one or more of the following: gemcitabine (GEMZAR®), vemurafenib (ZELBORAF®), dabrafenib (TAFINLAR®), trametinib (MEKINIST®), doxorubicin (ADRIAMYCIN®), cannefenib (BRAFTOVI®), cobimetinib (COTELLIC®), bimetinib (MEKTOVI®), dacarbazine (DTIC), temozolomide (TEMODAR®), ipilimumab (YERVOY®), pembrolizumab (KEYTRUDA®), nivolumab (OPDIVO®), interleukin (Proleukin®), recombinant interferon Alfa-2b (intron A), pegylated interferon Alfa-2b (PEG-intron / Sylatron), oxaliplatin and latamok (Talimogene Laherparepvec) (IMLYGIC®).

[0040] E28. A microneedle device or multiple microneedles according to any one of embodiments E1, E3-E5 or E7-E27, wherein the immunomodulator is selected from checkpoint inhibitors, Toll-like receptor (TLR) agonists, STING agonists, RIG agonists, cancer vaccines and cytokines.

[0041] E29. The microneedle device or multiple microneedles according to embodiment E28, wherein the checkpoint inhibitor inhibits a checkpoint molecule selected from the following: CTLA4, PD1, PD-L1, PD-L2, TIM3, LAG3, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), BTLA, KIR, MHC class I, MHC class II, GAL9, VISTA, BTLA, TIGIT, LAIR1, and A2aR.

[0042] E30. The microneedle device or multiple microneedles according to embodiment E28 or E29, wherein the checkpoint inhibitor is a PD-1 inhibitor.

[0043] E31. The microneedle device or multiple microneedles according to embodiment E28 or E29, wherein the checkpoint inhibitor is a CTLA4 inhibitor.

[0044] E32. The microneedle device or multiple microneedles according to embodiment E28, wherein the TLR agonist is selected from TLR-1 agonist, TLR-2 agonist, TLR-3 agonist, TLR-4 agonist, TLR-5 agonist, TLR-6 agonist, TLR-7 agonist, TLR-8 agonist, TLR-9 agonist, TLR-10 agonist, TLR-1 / 2 agonist, TLR-2 / 6 agonist, or TLR-7 / 8 agonist.

[0045] E33. The microneedle device or multiple microneedles according to embodiment E28 or E32, wherein the TLR agonist is a TLR-7 agonist.

[0046] E34. The microneedle device or multiple microneedles according to embodiment E28 or E32, wherein the TLR agonist is a TLR-9 agonist (e.g., unmethylated CG dinucleotide (CpG ODN)).

[0047] E35. The microneedle device or multiple microneedles according to embodiment E28, wherein the STING agonist is a cyclic dinucleotide, such as a cyclic dinucleotide containing a purine or pyrimidine nucleobase (e.g., adenosine, guanine, uracil, thymine, or cytosine nucleobase), optionally bis-(3'-5')-cyclic diguanosine monophosphate (c-di-GMP).

[0048] E36. The microneedle device or multiple microneedles according to embodiment E28, wherein the cytokines are GM-CSF, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-15, IL-21, IFN-α, IFN-β, IFN-γ, MIP-1α, MIP-1β, TGF-β, TNF-α, and TNFβ.

[0049] E37. The microneedle device or multiple microneedles according to embodiment E28, wherein the cytokines are GM-CSF, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-15, IL-18, IL-21, IFN-α, IFN-β, IFN-γ, MIP-1α, MIP-1β, TGF-β, TNF-α, or TNFβ.

[0050] E38. The microneedle device or multiple microneedles according to embodiment E28 or E36, wherein the cytokine is IL-2.

[0051] E39. The microneedle device or multiple microneedles according to embodiment E28 or E36, wherein the cytokine is IL-12.

[0052] E40. The microneedle device or multiple microneedles according to embodiment E28 or E36, wherein the cytokine is IL-15.

[0053] E41. The microneedle device or multiple microneedles according to embodiment E28 or E37, wherein the cytokine is IL-18.

[0054] E42. A microneedle device or multiple microneedles according to any one of embodiments E28, E36 or E37, wherein the cytokine is an engineered cytokine.

[0055] E43. A microneedle device or multiple microneedles according to any one of embodiments E28, E36 or E37, wherein the cytokine is an engineered interleukin (e.g., engineered IL-2 or IL-18).

[0056] E44. A microneedle device or multiple microneedles according to any one of embodiments E28, E36 or E37, wherein the cytokine is engineered interleukin-2.

[0057] E45. The microneedle device or multiple microneedles according to embodiment E28 or E37, wherein the cytokine is engineered interleukin-18.

[0058] E46. A microneedle device or multiple microneedles according to any one of embodiments E28, E36 or E37, wherein the cytokine is an anti-decoy interleukin.

[0059] E47. The microneedle device or multiple microneedles according to embodiment E28 or E37, wherein the cytokine is anti-decoy interleukin-18.

[0060] E48. The microneedle device or multiple microneedles according to embodiment E28 or E36, wherein the cytokine is GM-CSF.

[0061] E49. A microneedle device or multiple microneedles according to any of the foregoing embodiments, configured to administer anti-PD1 antibody and / or anti-CTLA4 antibody in combination with one or more of the following: (i) IL-2; (ii) IL-12; (iii) IL-15; (iv) Gemcitabine (GEMZAR®); (v) Vermupini (ZELBORAF®); (vi) Dabrafenib (TAFINLAR®); (vii) Trametinib (MEKINIST®); (viii) Dorothycin®; (ix) c-di-GMP; (x) mRNA; (xi) TLR-9 agonists (e.g., unmethylated CG dinucleotides (CpG ODN)); (xii) Oxaliplatin; and (xiii) GM-CSF.

[0062] E50. The microneedle device or multiple microneedles according to any one of the foregoing embodiments, configured to administer anti-PD1 antibody and / or anti-CTLA4 antibody in combination with one or more of the following: (i) IL-2; (ii) IL-12; (iii) IL-15; (iv) IL-18 (v) Gemcitabine (GEMZAR®); (vi) Vemurafenib (ZELBORAF®); (vii) Dabrafenib (TAFINLAR®); (viii) Trametinib (MEKINIST®); (ix) Dorothycin®; (x) c-di-GMP; (xi) mRNA; (xii) TLR-9 agonists (e.g., unmethylated CG dinucleotides (CpG ODN)); (xiii) Oxaliplatin; and (xiv) GM-CSF.

[0063] E51. The microneedle device or multiple microneedles according to embodiment E28 or E37, configured to administer anti-PD1 antibody and / or anti-CTLA4 antibody in combination with one or more of IL-2, IL-12 or IL-18.

[0064] E52. The microneedle device or multiple microneedles according to any one of the foregoing embodiments, configured to administer anti-PD1 antibody and / or anti-CTLA4 antibody in combination with IL-2 and IL-12.

[0065] E53. The microneedle device or multiple microneedles according to any one of the foregoing embodiments, configured to administer anti-PD1 antibody and / or anti-CTLA4 antibody in combination with IL-2 and TLR-9 agonists (e.g., unmethylated CG dinucleotide (CpG ODN)).

[0066] E54. The microneedle device or multiple microneedles according to any one of the foregoing embodiments, configured to administer IL-2 and c-di-GMP, optionally IL-12.

[0067] E55. The microneedle device or multiple microneedles according to any one of the foregoing embodiments, configured to administer IL-12 and c-di-GMP, optionally IL-2.

[0068] E56. A microneedle device or multiple microneedles according to any one of the foregoing embodiments, configured to administer a cancer vaccine, optionally wherein the cancer vaccine comprises a tumor antigen, such as a neoantigen.

[0069] E57. A microneedle, said microneedle comprising: (i) Substrate (e.g., a soluble substrate), (ii) An implantable silk fibroin tip comprising silk fibroin applied to or attached to a substrate, and (iii) (Optional) A backing applied to the substrate, The microneedles are configured to implant silk fibroin tips into the biological barrier (e.g., skin) of a subject, such as a human subject. The silk fibroin tip contains silk fibroin, such as regenerated silk fibroin and / or recombinant silk fibroin. The silk fibroin tip also contains an amount of anticancer agent sufficient to induce an anticancer response.

[0070] E58. A microneedle device or a plurality of microneedles comprising the microneedles according to embodiment E57, wherein, optionally, at least two of the plurality of microneedles contain the same anticancer agent or different anticancer agents.

[0071] E59. A microneedle, said microneedle comprising: (i) Substrate (e.g., a soluble substrate), (ii) An implantable silk fibroin tip comprising silk fibroin applied to a substrate, and (iii) (Optional) A backing applied to the substrate, The microneedles are configured to implant silk fibroin tips into the biological barrier (e.g., skin) of a subject, such as a human subject. The silk fibroin tip contains silk fibroin, such as regenerated silk fibroin and / or recombinant silk fibroin. The silk fibroin tip also contains an amount of immunomodulator sufficient to stimulate and / or suppress the immune system.

[0072] E60. A microneedle device or a plurality of microneedles according to any one of embodiments E1, E3-E5, E7-E56 or E59, optionally wherein at least two of the plurality of microneedles contain the same immunomodulator or different immunomodulators.

[0073] E61. A microneedle device comprising multiple silk fibroin-based microneedles, wherein the multiple microneedles comprise one or more microneedles according to any one of the foregoing embodiments.

[0074] E62. The microneedle device, multiple microneedles or microneedles according to any one of the foregoing embodiments, wherein the device, multiple microneedles or microneedles are configured to continuously release anticancer agents and / or immunomodulators.

[0075] E63. The microneedle device, multiple microneedles, or microneedles according to embodiment E62, wherein the sustained release comprises substantially continuous low-dose administration of the anticancer agent and / or the immunomodulator.

[0076] E64. The microneedle device, multiple microneedles, or microneedles according to embodiment E62 or E63, wherein the continuous release comprises approximately greater than 0% to approximately 100% of the total amount of anticancer agent and / or immunomodulator present in the silk fibroin tip.

[0077] E65. A microneedle device, a plurality of microneedles, or microneedles according to any one of embodiments E62-E64, wherein the continuous release is over a period of time including at least about 3 days (e.g., about 3, 4, 5, 6, 7 or more days, e.g., about 5 to about 10 days, e.g., about 7 to about 15 days, e.g., about 1 week to about 2 weeks, about 1 week to about 3 weeks, or about 2 weeks to about 4 weeks, e.g., about 1 month to about 3 months, e.g., about 2 months to about 4 months, e.g., about 3 months to about 6 months).

[0078] E66. A microneedle device, multiple microneedles or microneedles according to any one of embodiments E62-E65, wherein the continuous release is over a period of about 2 days to about 28 days.

[0079] E67. A microneedle device, multiple microneedles or microneedles according to any one of embodiments E62-E66, wherein the continuous release is over a period of about 5 days to about 21 days.

[0080] E68. The microneedle device, multiple microneedles, or microneedles according to any one of the foregoing embodiments, configured to release an effective amount of an anticancer agent and / or an immunomodulator to enhance the subject’s immune system exposure to cancer-associated neoantigens (e.g., neoantigens released after tumor cell lysis), thereby inducing and / or amplifying immune effector cells, such as T cells, specific to that neoantigen.

[0081] E69. The microneedle device, multiple microneedles or microneedles according to any one of the foregoing embodiments, configured to release an effective amount of anticancer agent and / or immunomodulator to induce T cell activation and / or overcome immunosuppression in the tumor microenvironment.

[0082] E70. A microneedle device, multiple microneedles or microneedles according to any one of the foregoing embodiments, wherein the device, multiple microneedles or microneedles are configured to release the anticancer agent and / or the immunomodulatory agent abruptly.

[0083] E71. The microneedle device, multiple microneedles, or microneedles according to embodiment E70, wherein the sudden release includes rapid administration of the anticancer agent and / or the immunomodulator.

[0084] E72. The microneedle device, multiple microneedles, or microneedles according to embodiment E70 or E71, wherein the sudden release comprises rapidly applying more than 0% to about 100% of the total amount of anticancer agent and / or immunomodulator present in the silk fibroin tip.

[0085] E73. A microneedle device, multiple microneedles or microneedles according to any one of embodiments E70-E72, wherein the sudden release is within a time period including at least about 1 hour (e.g., about 1 to about 30 minutes, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or 24 hours).

[0086] E74. A microneedle device, multiple microneedles or microneedles according to any one of embodiments E12, E15, E16 or E62-E73, wherein the release of the anticancer agent occurs at substantially the same rate (e.g., simultaneously) as the release of the immunomodulator.

[0087] E75. A microneedle device, multiple microneedles, or microneedles according to any one of embodiments E12, E15, E16, or E62-E74, wherein the release of the anticancer agent occurs at a different rate than the release of the immunomodulator, such that the anticancer agent is released substantially before or substantially after the release of the immunomodulator.

[0088] E76. The microneedle device, multiple microneedles or microneedles according to any one of the foregoing embodiments, configured to be applied (e.g., administered) to a biological barrier selected from the skin layer, cell membrane, mucous surface, oral cavity or buccal cavity.

[0089] E77. The microneedle device, multiple microneedles or microneedles according to any one of the foregoing embodiments, configured to be applied (e.g., administered) to a tumor (e.g., a metastatic tumor).

[0090] E78. The microneedle device, multiple microneedles or microneedles according to any one of the foregoing embodiments, configured to be applied (e.g., administered) to the tumor site after tumor resection, for example to induce an immune response to the tumor and / or ablate any cancer cells remaining after resection.

[0091] E79. The microneedle device, multiple microneedles or microneedles according to any one of the foregoing embodiments, configured to be applied (e.g., administered) to the tumor site prior to tumor resection, for example to induce an immune response to the tumor and / or to ablate any cancer cells remaining after resection.

[0092] E80. The microneedle device, multiple microneedles or microneedles according to any one of the foregoing embodiments, configured to be applied (e.g., administered) to the skin.

[0093] E81. The microneedle device, multiple microneedles or microneedles according to any one of the foregoing embodiments, configured to be applied (e.g., administered) to the eye.

[0094] E82. The microneedle device, multiple microneedles or microneedles according to any one of the foregoing embodiments, configured to be applied (e.g., administered) to the oral cavity.

[0095] E83. The microneedle device, multiple microneedles or microneedles according to any one of the foregoing embodiments, configured to be applied (e.g., administered) within a tumor.

[0096] E84. The microneedle device, multiple microneedles or microneedles according to any one of the foregoing embodiments, configured to be applied (e.g., administered) around a tumor.

[0097] E85. A microneedle device, multiple microneedles or microneedles according to any one of the foregoing embodiments, configured to be applied (e.g., administered) to a skin lesion or adjacent skin lesion (e.g., skin lesion associated with cancer or precancerous conditions).

[0098] E86. The microneedle device according to embodiment E12, wherein the local and / or systemic delivery (e.g., release) results in: (i) Inhibition of tumor growth at or near the application site; (ii) Induction of a local immune response to ablate tumors at or near the application site; (iii) An increase in activated immune effector cells (e.g., T cells) in the tumor microenvironment; (iv) Decrease in local immunosuppressive cells (e.g., regulatory T cells (Tregs)); (v) Induction of a systemic immune response to ablate tumors at distant sites; (vi) Immune memory of cancer or precancerous conditions; and / or (vii) Immune response to tumor antigens such as neoantigens; and / or (viii) Prevention and / or suppression of cancer recurrence (e.g., cancer relapse).

[0099] E87. A microneedle device, a plurality of microneedles, or microneedles according to any one of embodiments E4 or E57-E59, wherein the backing is selected from solid supports, such as paper-based materials, plastic materials, polymeric materials, or polyester-based materials (e.g., Whatman 903 paper, polymer tape, plastic tape, adhesive-backed polyester tape, or other medical tape).

[0100] E88. A microneedle device, a plurality of microneedles, or microneedles according to any one of embodiments E4, E9, E57-E59, or E87, wherein the substrate (e.g., a soluble substrate) comprises two or more of the following: (i) Polysaccharides (e.g., dextran); (ii) Disaccharides (e.g., sucrose, maltose, and trehalose); (iii) Polymers (e.g., methylcellulose, polyethylene glycol (PEG), carboxymethyl cellulose (CMC), polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), and hyaluronic acid salts); (iv) Proteins (e.g., gelatin); (v) Plasticizers (e.g., glycerin, propylene glycol); and (vi) Surfactants (e.g., octylphenol ethoxylates (e.g., Triton-X), polysorbates, poloxamer and / or polyethoxylated alcohols).

[0101] E89. A microneedle device, multiple microneedles, or microneedles according to any one of embodiments E4, E9, E57-E59, E87, or E88, wherein the substrate comprises one or more of the following: gelatin, dextran, glycerol, polyethylene glycol (PEG) (e.g., including low molecular weight PEG), sucrose, trehalose, maltose, carboxymethyl cellulose (CMC), polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), hyaluronic acid salt, methylcellulose, and / or surfactants (e.g., octylphenol ethoxylate (e.g., Triton-X), polysorbate, poloxamer such as P188, and / or polyethoxylated alcohols), optionally wherein the microneedles are configured for sustained release and / or sudden release.

[0102] E90. A microneedle device, a plurality of microneedles or microneedles according to any one of embodiments E4, E9, E57-E59 or E87-E89, wherein the substrate comprises dextran, sucrose, glycerol and a surfactant, optionally configured for sustained release.

[0103] E91. A microneedle device, multiple microneedles, or microneedles configured for continuous release according to any one of embodiments E62-E67, E89, or E90, comprising (optionally, a composition of a solution for casting and / or a dried, solidified substrate): (i) Approximately 20% to approximately 40%, for example, 30% of 70 kDa dextran; (ii) Approximately 5% to approximately 15%, for example, approximately 10% sucrose; (iii) Glycerin, about 0.5% to about 2.5%, for example about 1%; and (iv) About 0.001% to about 1%, for example about 0.01% of Triton-X.

[0104] E92. A microneedle device, a plurality of microneedles or microneedles according to any one of embodiments E88-E91, wherein the dextran has a molecular weight of about 30 kDa to about 600 kDa.

[0105] E93. A microneedle device, a plurality of microneedles, or microneedles according to any one of embodiments E88-E92, wherein the dextran is derived from... Leuconostoc mesenteroides .

[0106] E94. The microneedle device, multiple microneedles or microneedles of any one of the embodiments E4, E8, E9, E57-E59, E87 or E88-E93, wherein the substrate comprises polyvinyl alcohol (PVA) and sucrose, optionally configured for sudden release.

[0107] E95. A microneedle device, multiple microneedles, or microneedles configured for sudden release according to any one of embodiments E70-E72, E89, or E94, comprising (optionally, a composition of a solution for casting and / or a dried, solidified substrate): (i) PVA of approximately 15% to approximately 20%, for example, approximately 18%; (ii) Sucrose of about 25% to about 75%, for example about 50% sucrose; (iii) PVA of approximately 25% to approximately 75%, for example, approximately 50% PVA; and (iv) Approximately 15% to approximately 20%, for example, approximately 18% sucrose.

[0108] E96. A microneedle device, a plurality of microneedles or microneedles according to any one of embodiments E4, E8, E9, E57-E59, E87 or E88-E95, wherein the substrate does not contain poly(acrylic acid) (PAA).

[0109] E97. A microneedle device, a plurality of microneedles, or microneedles according to any one of embodiments E4, E8, E9-E96, wherein the silk fibroin tip comprises two or more of the following: (i) Disaccharides (e.g., sucrose, maltose and trehalose); (ii) Polymers (e.g., methylcellulose, polyethylene glycol (PEG), carboxymethyl cellulose (CMC), polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), hyaluronic acid salts); (iii) Amino acids (e.g., threonine); (iv) Plasticizers (e.g., glycerin, propylene glycol); and (v) Buffers (e.g., PBS).

[0110] E98. A microneedle device, a plurality of microneedles or microneedles according to any one of embodiments E4, E8, E9-E97, wherein the silk fibroin tip contains an excipient.

[0111] E99. A microneedle device, a plurality of microneedles or microneedles according to any one of embodiments E4, E8, E9-E98, wherein the silk fibroin tip comprises one or more of carboxymethyl cellulose (CMC), sucrose and threonine.

[0112] E100. A microneedle device, multiple microneedles or microneedles according to any one of embodiments E4, E8, E9-E99, wherein the silk fibroin tip contains a buffer, optionally phosphate-buffered saline (PBS).

[0113] E101. A microneedle device, a plurality of microneedles or microneedles according to any one of embodiments E70-E72, E89, E94 or E95, wherein the silk fibroin tip configured for sudden release contains about 2% to about 8% sucrose (e.g., about 5% sucrose).

[0114] E102. A microneedle device, a plurality of microneedles or microneedles according to any one of embodiments E70-E72, E89, E94, E95 or E101, wherein the silk fibroin tip configured for burst release contains about 0.5% to about 3% w / v CMC (e.g. about 1% CMC).

[0115] E103. A microneedle device, a plurality of microneedles or microneedles according to any one of embodiments E70-E72, E89, E94, E95, E101 or E102, wherein the silk fibroin tip configured for sudden release contains about 50 mM to about 100 mM of amino acids, such as threonine (e.g., about 75 mM of threonine).

[0116] E104. A microneedle device, a plurality of microneedles, or microneedles according to any one of embodiments E4, E8, or E9-E103, wherein the silk fibroin tip comprises about 1% w / v to about 10% w / v (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% w / v) of silk fibroin, or has according to Figure 5 Silk fibroin with a molecular weight distribution, or silk fibroin containing, for example, about 20 μg to about 245 μg per 121 microneedle arrays.

[0117] E105. A microneedle device, a plurality of microneedles, or microneedles according to any one of embodiments E4, E8, or E9-E103, wherein the silk fibroin tip comprises a 10 MB silk fibroin solution of about 1% w / v to about 10% w / v (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% w / v), or according to Figure 5 Silk fibroin solution.

[0118] E106. A microneedle device, a plurality of microneedles, or microneedles according to any one of embodiments E4, E8, or E9-E103, wherein the silk fibroin tip comprises a 60 MB silk fibroin solution of about 1% w / v to about 10% w / v (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% w / v), or according to Figure 5 Silk fibroin solutions, for example, 100kDa to 200kDa (e.g., about 153kDa) silk fibroin solutions.

[0119] E107. A microneedle device, a plurality of microneedles, or microneedles according to any one of embodiments E4, E8, or E9-E103, wherein the silk fibroin tip comprises a 120 MB silk fibroin solution of about 1% w / v to about 10% w / v (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% w / v), or according to Figure 5 Silk fibroin solutions, for example, 70kDa to 150kDa (e.g., about 100kDa) silk fibroin solutions.

[0120] E108. A microneedle device, a plurality of microneedles, or microneedles according to any one of embodiments E4, E8, or E9-E103, wherein the silk fibroin tip comprises a 180MB silk fibroin solution of about 1% w / v to about 10% w / v (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% w / v), or according to Figure 5 Silk fibroin solutions, for example, 36kDa to 100kDa (e.g., about 71kDa) silk fibroin solutions.

[0121] E109. A microneedle device, a plurality of microneedles, or microneedles according to any one of embodiments E4, E8, or E9-E103, wherein the silk fibroin tip comprises a 480MB silk fibroin solution of about 1% w / v to about 10% w / v (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% w / v), or according to Figure 5 Silk fibroin solutions, for example, 1 kDa to 60 kDa (e.g., about 16 kDa) silk fibroin solutions.

[0122] E110. A microneedle device, multiple microneedles or microneedles according to any one of embodiments E62-E67, E89, E90 or E91, wherein the silk fibroin tip configured for continuous release contains about 1% to about 10% w / v of 60MB silk fibroin solution.

[0123] E111. A microneedle device, multiple microneedles or microneedles according to any one of embodiments E62-E67, E89, E90 or E91, wherein the silk fibroin tip configured for continuous release contains about 1% to about 10% w / v of 60MB silk fibroin solution.

[0124] E112. A microneedle device, a plurality of microneedles or microneedles according to any one of embodiments E62-E67, E89, E90 or E91, wherein the silk fibroin tip configured for continuous release comprises about 1% to about 10% w / v of 120MB silk fibroin solution.

[0125] E113. A microneedle device, a plurality of microneedles or microneedles according to any one of embodiments E62-E67, E89, E90 or E91, wherein the silk fibroin tip configured for continuous release comprises about 1% to about 10% w / v of 180MB silk fibroin solution.

[0126] E114. A microneedle device, multiple microneedles or microneedles according to any one of embodiments E62-E67, E89, E90 or E91, wherein the silk fibroin tip configured for continuous release contains about 1% to about 10% w / v of 480MB silk fibroin solution.

[0127] E115. The microneedle device, multiple microneedles or microneedles according to any one of the foregoing embodiments, which substantially contains only soluble materials, such as substantially only polymer-based and / or sugar-based materials.

[0128] E116. The microneedle device, multiple microneedles or microneedles according to any one of the foregoing embodiments, configured to be applied to a tumor site, for example after resection, and left in place to completely dissolve.

[0129] E117. A microneedle device, multiple microneedles, or microneedles according to any one of embodiments E4, E8, or E9-E114, wherein the microneedles are configured to implant the silk fibroin tip into the subject's biological barrier at a depth of about 100 μm to about 1 mm (e.g., the maximum penetration depth at the distal end of the tip).

[0130] E118. The microneedle device, multiple microneedles or microneedles according to any one of the foregoing embodiments, wherein the length of the microneedles is between about 350 μm and about 1500 μm.

[0131] E119. A microneedle device, a plurality of microneedles or microneedles according to any one of embodiments E4, E8, E9-E114 or E117, wherein the height of the silk fibroin tip may extend to approximately half of the total height of the microneedle.

[0132] E120. A microneedle device, a plurality of microneedles or microneedles according to any one of embodiments E4, E8, E9-E114 or E117, wherein the height of the silk fibroin tip is between about 75 µm and about 475 µm.

[0133] E121. A microneedle device, a plurality of microneedles or microneedles according to any one of embodiments E4, E8, E9-E114 or E117, wherein the silk fibroin tip has a tip radius of about 0.5 μm to about 100 μm.

[0134] E122. A microneedle device, a plurality of microneedles or microneedles according to any one of embodiments E4, E8, E9-E114 or E117, wherein the silk fibroin tip has a tip radius of about 5 μm to about 10 μm.

[0135] E123. A microneedle device, a plurality of microneedles or microneedles according to any one of embodiments E4, E8, E9-E114 or E117, wherein the silk fibroin tip comprises an angle of about 5 degrees to about 45 degrees.

[0136] E124. A method for treating cancer (e.g., metastatic cancer) and / or inducing an immune response to cancer (e.g., metastatic cancer), comprising bringing a microneedle device or a plurality of microneedles according to any one of embodiments E1-E56, E58, or E60-E123 into contact (e.g., application) a cancer (e.g., metastatic tumor) site of a subject, thereby producing one or more of the following: (i) Lysing cancer cells, such as tumor cells, to release cancer-associated antigens (e.g., neoantigens) and / or exposing cancer-associated antigens (e.g., neoantigens) to the subject's immune system; (ii) Cancer-associated antigens (e.g., neoantigens) that are complexed with the major histocompatibility complex (MHC) are presented on the surface of immune system cells (e.g., helper cells such as B cells, dendritic cells, etc.) by antigen-presenting cells (APCs). (iii) Cancer-associated antigens (e.g., neoantigens) that are recognized by immune effector cells (e.g., T cells and / or NK cells); (iv) Activating and / or expanding immune effector cells, such as T cells and / or NK cells, that are specific for cancer-associated antigens (e.g., neoantigens) displayed in the subject; and (v) Enhance (e.g., stimulate or upregulate) the immune response of immune effector cells such as T cells and / or NK cells, which promote the killing of target cells expressing cancer-associated antigens (e.g., neoantigens) in the subject and / or inhibit the growth or proliferation of said target cells.

[0137] E125. The method according to embodiment E124, wherein the cancer expresses multiple antigens (e.g., neoantigens), but wherein the tumor microenvironment inhibits the activation of immune effector cells (e.g., T cells) that recognize them.

[0138] E126. The method according to embodiment E124 or E125, wherein an enhanced (e.g., stimulated or upregulated) immune response to target cells expressing cancer-associated antigens (e.g., neoantigens) occurs at or near the application site.

[0139] E127. The method according to any one of embodiments E124-E126, wherein the enhanced (e.g., stimulated or upregulated) immune response to target cells expressing cancer-associated antigens (e.g., neoantigens) is a systemic immune response (e.g., a broad-spectrum response) to ablate tumors at distant sites.

[0140] E128. A method for treating cancer (e.g., metastatic cancer) and / or inducing an immune response to cancer (e.g., metastatic cancer), comprising bringing a microneedle device or a plurality of microneedles according to any one of embodiments E1-E56, E58 or E60-E123 into contact (e.g., application) a cancer (e.g., metastatic tumor) site of a subject.

[0141] E129. A method of treating cancer (e.g., metastatic cancer) or precancerous conditions (e.g., precancerous skin conditions), comprising bringing a microneedle device or a plurality of microneedles according to any one of embodiments E1-E56, E58 or E60-E123 into contact (e.g., application) a tumor (e.g., metastatic tumor) or lesion (e.g., skin lesion) of a subject.

[0142] E130. A method for preventing cancer recurrence, comprising bringing a microneedle device or multiple microneedles according to any one of embodiments E1-E56, E58 or E60-E123 into contact (e.g., application) a tumor (e.g., metastatic tumor, such as on the skin) of a subject.

[0143] E131. A method of treating cancer (e.g., metastatic cancer) comprising bringing a microneedle device or a plurality of microneedles according to any one of embodiments E1-E56, E58 or E60-E123 into contact (e.g., application) a surgical resection site or a proximal site of a subject.

[0144] E132. A method of treating cancer (e.g., metastatic cancer) comprising bringing a microneedle device or plurality of microneedles according to any one of embodiments E1-E56, E58 or E60-E123 into contact (e.g., application) with a tumor (e.g., metastatic tumor) site of a subject, thereby inducing: (i) Local immune response to cancer and / or local cytotoxicity (e.g., evidenced by death of cancer cells at or near the site of microneedling application, such as reduction in local tumor size and / or local tumor burden); and / or (ii) Distal immune response to cancer and / or distal cytotoxicity (e.g., as demonstrated by the death of cancer cells at a location distal to where the microneedle patch was applied, such as a reduction in distal tumor size and / or overall tumor burden).

[0145] E133. The method according to any one of embodiments E124-E132, wherein the contact (e.g., application) results in one or more of the following: (i) Inhibition of tumor growth at or near the application site; (ii) Induction of a local immune response to ablate tumors at or near the application site; (iii) An increase in activated immune effector cells (e.g., T cells) in the tumor microenvironment; (iv) Decrease in local immunosuppressive cells (e.g., regulatory T cells (Tregs)); (v) Induction of a systemic immune response to ablate tumors at distant sites; (vi) Immune memory of cancer or precancerous conditions; (vii) Immune response to tumor antigens such as neoantigens; and / or (viii) Prevention and / or suppression of cancer recurrence (e.g., cancer relapse).

[0146] E134. The method according to embodiment E133, wherein the immune memory results in the ablation of recurrent tumors at or near the application site and / or the ablation of recurrent tumors at a distant site, optionally when the recurrent tumor occurs within about 1 to about 6 months after application, optionally when the recurrent tumor occurs within about 1 to about 5 years after application.

[0147] E135. The method according to implementation scheme E133 or E134, wherein the immune memory prevents tumor recurrence.

[0148] E136. The method according to any one of embodiments E133-E135, wherein the immune memory prevents cancer recurrence within the first 5 years after the initial application of microneedles.

[0149] E137. The method according to any one of embodiments E124-E136, wherein the cancer is a metastatic cancer.

[0150] E138. The method according to any one of embodiments E124-E137, wherein the cancer is a recurrent cancer.

[0151] E139. The method according to any one of embodiments E124-E138, wherein the cancer is a refractory cancer.

[0152] E140. According to the method of any one of embodiments E124-E139, the cancer is selected from anal cancer; basal cell carcinoma; bladder cancer; bone cancer; brain tumor; breast cancer; cervical cancer; colon and rectal cancer; endometrial cancer; esophageal cancer; gastrointestinal stromal tumor; gestational trophoblastic disease; head and neck cancer; Hodgkin lymphoma; Kaposi's sarcoma; kidney (renal cell) carcinoma; leukemia; liver cancer; lung cancer; malignant mesothelioma; melanoma; Merkel cell carcinoma; multicentric Castleman's disease; multiple myeloma and other plasma cell tumors; myeloproliferative neoplasms; neuroblastoma; non-Hodgkin lymphoma; ovarian cancer, fallopian tube cancer, or primary peritoneal cancer; pancreatic cancer; penile cancer; pheochromocytoma and paraganglioma; prostate cancer; retinoblastoma; rhabdomyosarcoma; skin cancer; squamous cell carcinoma; soft tissue sarcoma; solid tumors anywhere in the body; gastric (stomach) cancer; testicular cancer; thyroid cancer; vaginal cancer; vulvar cancer; and Wilms' tumor and other pediatric kidney cancers.

[0153] E141. The method according to any one of embodiments E124-E140, wherein the cancer is melanoma.

[0154] E142. The method according to any one of embodiments E124-E140, wherein the cancer is basal cell carcinoma.

[0155] E143. The method according to any one of embodiments E124-E140, wherein the cancer is squamous cell carcinoma.

[0156] E144. The method according to any one of embodiments E124-E140, wherein the cancer is Merkel cell carcinoma.

[0157] E145. The method according to any one of embodiments E124-E140, wherein the cancer is breast cancer.

[0158] E146. The method according to any one of embodiments E124-E140, wherein the cancer is associated with skin lesions and / or tumors.

[0159] E147. The method according to any one of embodiments E124-E140, wherein the tumor is a metastatic tumor.

[0160] E148. The method according to any one of embodiments E124-E140, wherein the tumor is palpable without surgery.

[0161] E149. The method according to any one of embodiments E124-E140, wherein the tumor is surgically accessible.

[0162] E150. The method according to any one of embodiments E124-E140, wherein the tumor is on the skin.

[0163] E151. The method according to any one of embodiments E124-E140, wherein the tumor is on the eye.

[0164] E152. The method according to any one of embodiments E129 or E133-E151, wherein the precancerous condition is a precancerous skin condition, optionally selected from actinic keratosis (AK), malignant freckle-like nevus, leukoplakia of the mucosa and Bowen's disease.

[0165] E153. The method according to any one of embodiments E124-E152, wherein the contact (e.g., application) is performed within a tumor.

[0166] E154. The method according to any one of embodiments E124-E152, wherein the contact (e.g., application) is performed around the tumor.

[0167] E155. The method according to any one of embodiments E124-E152, wherein the contact (e.g., application) is performed prior to surgical resection.

[0168] E156. The method according to any one of embodiments E124-E152, wherein the contact (e.g., application) is performed after surgical excision.

[0169] E157. The method according to any one of embodiments E124-E152, wherein the contact (e.g., application) is performed simultaneously with the collection of surgical and / or biopsy samples.

[0170] E158. The method according to any one of embodiments E124-E152, wherein the contact (e.g., application) is combined with standard care treatment (e.g., standard care treatment for cancer or precancerous conditions), the standard care treatment optionally selected from surgery, chemotherapy, immunotherapy, targeted therapy, hormone therapy and / or radiation therapy.

[0171] E159. The method according to implementation scheme E158, wherein the standard care treatment is performed before, after, or simultaneously with the microneedle device.

[0172] E160. The method according to any one of embodiments E124-E159, wherein the subject is a human subject.

[0173] E161. A method of manufacturing a microneedle device, the method comprising: A mold is provided comprising a mold body having an array of needle cavities of predetermined shapes formed therein, such as pyramidal and / or conical needle cavities; The tip of the needle cavity is filled with a composition comprising a solution of silk fibroin, an anticancer agent, and / or an immunomodulator. The filling tip of the needle cavity is dried to produce a silk fibroin tip, and the needle tip is optionally annealed. The needle cavity of the mold is filled with a base (e.g., a soluble base) solution; Dry the substrate solution to produce a base layer for the silk fibroin tip; and (Optionally) A backing is applied to the substrate layer to fabricate a microneedle device.

[0174] E162. A method of manufacturing a microneedle device, the method comprising: A mold is provided comprising a mold body having an array of needle cavities of predetermined shapes formed therein, such as pyramidal and / or conical needle cavities; The tip of the needle cavity is filled with a composition comprising a solution of silk fibroin and a therapeutic agent (e.g., an anticancer agent, an immunomodulator, or both); The filling tip of the needle cavity is dried to produce a silk fibroin tip, and the silk fibroin tip is optionally annealed. The needle cavity of the mold is further filled with a first base (e.g., a soluble base) solution; The first substrate solution is dried to form a first substrate layer; (Optionally) One or more additional base layers are formed by adding one or more additional base solutions to a first base layer and drying the additional base solutions, wherein the additional base solutions are different from the first base solution. (Optionally) A backing is applied to a substrate layer (e.g., a first substrate layer, or one or more other substrate layers) to fabricate a microneedle device.

[0175] E163. The method according to embodiment E161 or E162, wherein the base solution (e.g., a first base solution, one or more other base solutions, or both) comprises a molten liquid.

[0176] E164. The method according to embodiment E161 or E162, wherein the base solution (e.g., a first base solution, one or more other base solutions, or both) comprises a slurry.

[0177] E165. The method according to any one of embodiments E161-E163, wherein filling (e.g., a mold or needle cavity) comprises filling with a base solution containing molten liquid.

[0178] E166. The method according to any one of embodiments E161-E165, wherein filling (e.g., mold or needle cavity) comprises filling with a base solution containing slurry.

[0179] E167. The method according to any one of embodiments E161-E166 further includes using a chemical reaction (e.g., after filling) to solidify the base layer (e.g., a first base layer, one or more additional base layers, or both).

[0180] E168. The method according to any one of embodiments E161-E167, further comprising removing the microneedle device from the mold, optionally before applying the backing.

[0181] E169. The method according to any one of embodiments E161-E168, wherein the microneedle device is removed by bending the mold away from the microneedle device.

[0182] E170. The method according to any one of embodiments E161-E169 further includes packaging the microneedle device in a container having a low water vapor permeability, the container having a desiccant to maintain a relative humidity of about 0% to about 50% (e.g., about 0% to 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, or about 40% to 50%, such as about 25%) within the package.

[0183] E171. The method according to any one of embodiments E161-E170, wherein silk fibroin, anticancer agent and / or immunomodulator solution are dispensed into each needle cavity in the mold via nano-printing.

[0184] E172. The method according to any one of embodiments E161-E171, wherein filling the tip of the needle cavity comprises dispensing a solution, such as a silk fibroin, anticancer agent, and / or immunomodulatory agent solution, into each needle cavity.

[0185] E173. The method according to any one of embodiments E161-E172, wherein drying the filling tip of the needle cavity includes a primary drying step and a secondary drying step.

[0186] E174. The method according to any one of embodiments E161-E173, wherein drying the substrate (e.g., soluble substrate) solution comprises subjecting the mold to centrifugation at 3900 rpm for 2 minutes and topping off the needle cavity with 50 μL of substrate solution.

[0187] E175. The method according to any one of embodiments E161-E174, wherein the substrate filling is performed by nanoliter (nL) distribution (e.g., nanoliter printing).

[0188] E176. The method according to any one of embodiments E161-E175 further includes an annealing step after filling the tip of the needle cavity (e.g., before filling the substrate).

[0189] E177. The method according to any one of embodiments E161-E176 further includes a water annealing step after filling the tip of the needle cavity (e.g., before filling the substrate).

[0190] E178. The method according to any one of embodiments E161-E177, wherein the backing layer comprises one of a paper backing layer and an adhesive plastic tape.

[0191] E179. The method according to any one of embodiments E161-E178, wherein the backing layer comprises an adhesive-coated plastic tape.

[0192] E180. The method according to any one of embodiments E161-E179, wherein the backing layer comprises a porous layer.

[0193] E181. The method according to any one of embodiments E161-E180, wherein the backing layer comprises one or more adhesives selected from the group consisting of acrylic acid, acrylate, cyanoacrylate, silicone, polyurethane and synthetic rubber.

[0194] E182. The method according to any one of embodiments E161-E181, wherein the backing layer comprises an adhesive capable of curing by light irradiation.

[0195] E183. A microneedle device, plurality of microneedles, microneedles, or method according to any one of embodiments E1-E182, wherein the microneedle device, plurality of microneedles, microneedles, or components thereof contain silk fibroin in an amount of about 0.5 µg to about 500 µg (e.g., about 0.5 µg to about 5 µg, or about 1 µg to about 10 µg, or about 5 µg to about 15 µg, or about 10 µg to about 20 µg, or about 15 µg to about 25 µg, or about 20 µg to about 30 µg, or about 25 µg to about 35 µg, or about 30 µg to about 40 µg, or about 35 µg to about 45 µg, or about 40 µg to about 50 µg, or about 45 µg to about 55 µg, or about 50 µg to about 60 µg, or about 55µg to about 65µg, or about 60µg to about 70µg, or about 65µg to about 75µg, or about 70µg to about 80µg, or about 75µg to about 85µg, or about 80µg to about 90µg, or about 85µg to about 95µg, or about 90µg to about 100µg, or about 95µg to about 150µg, or about 125µg to about 175µg, or about 150µg to about 200µg, or about 225µg to about 275µg, or about 250µg to about 300µg, or about 325µg to about 375µg, or about 350µg to about 400µg, or about 425µg to about 475µg, or about 450µg to about 500µg of silk fibroin.

[0196] E184. A microneedle device, plurality of microneedles, microneedles or method according to any one of embodiments E1-E183, wherein the microneedle device, plurality of microneedles, microneedles or components thereof contain silk fibroin in an amount of about 1% to about 75% by weight (e.g. about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75% by weight of silk fibroin). Attached Figure Description

[0197] Figure 1 This is a schematic diagram of a microneedle manufacturing process according to an example of this disclosure.

[0198] Figure 2 This disclosure describes a series of microneedles configured, according to one example, to release different therapeutic agents either through sustained release or through abrupt (referred to as a "bolus") release. The leftmost microneedle is configured to release an anticancer agent, such as a chemotherapy agent, over a two-week period. The middle microneedle is configured to release an immunomodulatory agent, such as a checkpoint inhibitor (e.g., an anti-PD1 antibody), over a two-week period. The rightmost microneedle is configured to rapidly release an immunomodulatory agent, such as a cytokine (e.g., IL-2), over a short period of time, such as within minutes.

[0199] Figure 3 A complete microneedle device having an array of microneedles applied to a backing or "handle" layer is illustrated according to an example of this disclosure.

[0200] Figure 4 A microneedle device according to an example of this disclosure is illustrated. The microneedle device comprises multiple microneedles having sufficient mechanical properties (e.g., strength) and suitable geometry (e.g., tip sharpness, tip angle, length, and interneedle spacing) to pierce biological barriers (e.g., skin) to achieve local and / or systemic delivery of a therapeutic agent or combination of therapeutic agents (e.g., anticancer agents, immunomodulators, or combinations thereof) to a subject.

[0201] Figure 5 Various molecular weight distributions of silk fibroin solutions that can be used to manufacture the microneedles described herein are illustrated.

[0202] Figure 6A The dosing regimens for comparing intratumoral (IT) bolus administration of IL-2 or gemcitabine versus daily IT administration are illustrated in an exemplary mouse model. Figure 6B The graph depicts the change in tumor volume over time in mice treated with bolus IL-2 (IT) or daily IL-2 (IT). Figure 6C The graph shows the survival of mice treated with bolus injection (IT) or daily IL-2 (IT). Figure 6D The graph depicts the change in tumor volume over time in mice treated with bolus gemcitabine (IT) or daily gemcitabine (IT). Figure 6E The graph depicts survival in mice treated with either bolus injection (IT) or daily gemcitabine (IT).

[0203] Figures 7A-7B This illustrates the use of intratumoral bolus (IT) injection of gemcitabine (Gemcitabine) in an exemplary mouse model. Figure 7A ) and Daily IT Giscitabine ( Figure 7B Dosing regimen; Figure 7C The graph depicts the change in tumor volume over time in mice treated with bolus (IT) or daily (IT) gemcitabine. Figure 7D The graph depicts survival in mice treated with either bolus (IT) or daily (IT) gemcitabine.

[0204] Figure 8 The graph depicts the stability of IL-2 in exemplary silk formulations over a 14-day period at 4°C, room temperature (RT), or 37°C, as determined by IL-2 recovery (%). Detailed Implementation

[0205] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0206] The articles “a / an” and “a kind” are used in this text to refer to one or more (i.e., at least one) grammatical objects of the article. For example, “a element” refers to one element or more elements.

[0207] When referring to measurable values ​​such as quantity, duration, etc., the term “about” is intended to cover variations of ±20%, or in some cases ±10%, or in some cases ±5%, or in some cases ±1%, or in some cases ±0.1% based on the specified value, as such variations are suitable for, for example, implementing the disclosed method.

[0208] The terms "combination" or "in combination with" are not intended to suggest that therapies or treatments can be administered simultaneously and / or formulated for delivery together, but such delivery methods are within the scope described herein. A treatment in a combination may be administered simultaneously, before, or after one or more other additional therapies or treatments. Treatments or treatment regimens may be administered in any order. Typically, each agent will be administered at a dose and / or schedule determined for that agent. It should also be understood that additional treatments used in this combination may be administered together in a single composition or separately in different compositions. Generally, additional treatments used in combination are intended to be used at levels not exceeding those used individually. In some embodiments, the level used in combination will be lower than the level used individually.

[0209] The terms “inhibition,” “inhibitor,” or “antagonist” refer to a reduction in a parameter (e.g., activity) of a given molecule (e.g., an immune checkpoint inhibitor). For example, the term includes inhibition of activity (e.g., PD-1 or PD-L1 activity) by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more. Therefore, inhibition need not be 100%.

[0210] The terms “activator,” “activator,” or “agonist” refer to an increase in a parameter (e.g., activity) of a given molecule (e.g., a co-stimulatory molecule). For example, the term includes an increase in activity (e.g., co-stimulatory activity) of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more.

[0211] The terms "antitumor effect" and "anticancer effect" are used interchangeably and refer to biological effects that can be manifested by various means, including but not limited to, reduction of tumor or cancer volume, reduction of the number of tumor or cancer cells, reduction of the number of metastases, increase of life expectancy, reduction of tumor or cancer cell proliferation, reduction of tumor or cancer cell survival, prevention of recurrence, or improvement of various physiological symptoms associated with cancer. "Antitumor effect" or "anticancer effect" can also manifest as the ability of the microneedles of this disclosure to prevent the first occurrence of tumors or cancer and / or cancer recurrence or relapse. "Antitumor effect" or "anticancer effect" can also manifest as the ability of the microneedles of this disclosure to induce an immune response against cancer, for example, the ability to induce an immune response against cancer-associated antigens (e.g., neoantigens).

[0212] As used herein, "tumor antigen" or the interchangeable "cancer antigen" includes any molecule present on or associated with cancer, such as cancer cells or the tumor microenvironment that can elicit an immune response. As used herein, "immune cell antigen" includes any molecule present on or associated with immune cells that can elicit an immune response. In some embodiments, tumor antigen refers to a neoantigen (e.g., an antigen derived from somatic mutations in a tumor that differs from wild-type antigens and is specific to each tumor and / or subject, such as a peptide).

[0213] As used herein, the term "anticancer agent" refers to a therapy and / or drug that can induce antitumor and / or anticancer effects. In some embodiments, anticancer effects include, but are not limited to, reduction of tumor volume or cancerous volume, reduction of the number of tumor cells or cancer cells, reduction of the number of metastases, increase of life expectancy, reduction of tumor cell proliferation or cancer cell proliferation, reduction of tumor cell survival or cancer cell survival, prevention of recurrence, or improvement of various physiological symptoms associated with the cancer condition.

[0214] As used herein, an “increase” or “decrease” in a measurement is generally compared to a baseline value, unless otherwise stated. For example, an increase or decrease in a measurement may be compared to a baseline level expected in a healthy subject. Alternatively, an increase or decrease in a measurement may be compared to a previous time point in the same subject, such as before treatment. In some cases, an increase or decrease in a measurement may be compared to a previous time point in the same subject, such as during treatment.

[0215] As used herein, the term "immunomodulator" refers to a therapy and / or medicine that modulates (e.g., increases and / or decreases), enhances, induces, stimulates, inhibits, reduces, or upregulates one or more aspects of an immune response in a subject, such as someone with cancer. For example, immunomodulators can enhance or promote the immune attack of target cells, such as cancer cells, and / or promote the killing of target cells, such as cancer cells, by immune effector cells, or inhibit the growth or proliferation of target cells, such as cancer cells. In some embodiments, immunomodulators, as described herein, administered, for example, via microneedles or devices described herein, can enhance a subject's immune response to cancer.

[0216] As used herein, the term "cancer" is intended to include all types of cancerous growth or carcinogenic processes, metastatic tissue, or malignantly transformed cells, tissues, or organs, regardless of histopathological type or stage of invasion. Cancer cells can spread locally or to other parts of the body via the bloodstream and lymphatic system. Examples of cancerous conditions include, but are not limited to, solid tumors, blood cancers, soft tissue tumors, and metastatic lesions. Examples of solid tumors include malignancies of various organ systems, such as sarcomas and carcinomas (including adenocarcinomas and squamous cell carcinomas), such as those affecting the liver, lungs, breast, lymph nodes, gastrointestinal tract (e.g., colon), genitourinary tract (e.g., kidneys, urethral epithelial cells), prostate, and pharynx. Adenocarcinomas include malignancies such as most colon cancers, rectal cancers, renal cell carcinomas, liver cancers, non-small cell lung cancers, small bowel cancers, and esophageal cancers. Squamous cell carcinomas include malignancies such as those affecting the lungs, esophagus, skin, head and neck region, oral cavity, anus, and cervix. The methods and microneedles of this disclosure may also be used to treat or prevent metastatic lesions of the aforementioned cancers.

[0217] The terms “tumor” and “cancer” are used interchangeably in this document; for example, both terms cover solid tumors and liquid tumors, such as diffuse or circulating tumors. As used herein, the terms “cancer” or “tumor” include both pre-malignant and malignant cancers and tumors.

[0218] The term "antigen-presenting cell" or "APC" refers to immune system cells, such as helper cells (e.g., B cells, dendritic cells, etc.), that display exogenous antigens complexed with the major histocompatibility complex (MHC) on their surface. T cells can recognize these complexes using their T-cell receptors (TCRs). APCs process the antigens and present them to T cells.

[0219] As used herein, “immune cell” refers to any of the various cells that function in the immune system to, for example, protect against infectious agents and foreign substances. In embodiments, the term includes leukocytes, such as neutrophils, eosinophils, basophils, lymphocytes, and monocytes. Innate leukocytes include phagocytes (e.g., macrophages, neutrophils, and dendritic cells), mast cells, eosinophils, basophils, and natural killer cells. Innate leukocytes recognize and eliminate pathogens by attacking larger pathogens upon contact or by phagocytosis and then killing microorganisms, and are mediators in the activation of adaptive immune responses. Cells of the adaptive immune system are a special type of leukocyte called lymphocytes. B cells and T cells are important lymphocyte types and originate from hematopoietic stem cells in the bone marrow. B cells participate in humoral immune responses, while T cells participate in cell-mediated immune responses. The term “immune cell” also includes immune effector cells.

[0220] As used herein, the term "immune effector cell" or "effect cell" refers to a cell that participates in an immune response, such as promoting an immune effector response. Examples of immune effector cells include T cells, such as α / β T cells and γ / δ T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and bone marrow-derived phagocytes.

[0221] As used herein, the terms “immune effector” or “effect,” “function” or “response” refer to a function or response that enhances or promotes the immune attack of target cells, such as the function or response of immune effector cells. For example, an immune effector function or response refers to the property of T or NK cells to promote the killing of target cells or inhibit the growth or proliferation of target cells. In the case of T cells, primary stimulation and co-stimulation are examples of immune effector functions or responses.

[0222] The term "effective function" refers to a specialized function of a cell. The effector functions of T cells may include, for example, cytolytic activity or helper cell activity, including the secretion of cytokines.

[0223] As used herein, the term "treat / treatment / treating" refers to a reduction or improvement in the progression, severity, and / or duration of a condition (e.g., a proliferative condition) or an improvement in one or more symptoms of the condition (preferably, one or more identifiable symptoms) caused by the application of one or more therapies. In specific embodiments, the term "treat / treatment / treating" refers to improving at least one measurable physical parameter of a proliferative condition, such as tumor growth, which is not necessarily identifiable by the patient. In other embodiments, the term "treat / treatment / treating" refers to suppressing the progression of a proliferative condition physically by, for example, stabilizing identifiable symptoms, or physiologically by, for example, stabilizing physical parameters, or both. In other embodiments, the term "treat / treatment / treating" refers to reducing or stabilizing tumor size or cancer cell count.

[0224] The terms “polypeptide,” “peptide,” and “protein” (if single-chain) are used interchangeably herein and refer to polymers of amino acids of any length. These polymers may be linear or branched, may contain modified amino acids, and may be broken down by non-amino acid components. The term also covers polymers of modified amino acids; for example, those subjected to disulfide bond formation, glycosylation, esterification, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeled component. Polypeptides may be isolated from natural sources, produced from eukaryotic or prokaryotic hosts via recombinant technologies, or may be products of synthetic processes.

[0225] The terms “nucleic acid,” “nucleic acid sequence,” “nucleotide sequence,” or “polynucleotide sequence” and “polynucleotide” are used interchangeably. They refer to a polymeric form of nucleotides of any length, which are deoxyribonucleotides or ribonucleotides or analogues thereof. Polynucleotides can be single-stranded or double-stranded, and if single-stranded, can be coding or non-coding (antisense) strands. Polynucleotides may contain modified nucleotides, such as methylated nucleotides and nucleotide analogues. The sequence of nucleotides may be broken down by non-nucleotide components. Polynucleotides can be further modified after polymerization, such as by conjugation with labeled components. Nucleic acids can be recombinant polynucleotides, or genomic, cDNA, semi-synthetic, or synthetically derived polynucleotides that do not exist in nature or are linked to another polynucleotide in a non-natural arrangement.

[0226] In the context of nucleotide sequences, the term “substantially identical” is used herein to refer to a first nucleic acid sequence containing a sufficient or minimum number of nucleotides identical to the aligned nucleotides in a second nucleic acid sequence, such that the first and second nucleotide sequences encode polypeptides with common functional activities, or encode common structural polypeptide domains or common functional polypeptide activities, for example, nucleotide sequences having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with a control sequence (e.g., the sequence provided herein).

[0227] The term "variant" refers to a polypeptide having an amino acid sequence substantially identical to that of a control, or encoded by a substantially identical nucleotide sequence. In some embodiments, the variant is a functional variant.

[0228] The term "functional variant" refers to a polypeptide that has an amino acid sequence substantially identical to or encoded by a substantially identical nucleotide sequence and is capable of having one or more activities of the control amino acid sequence.

[0229] The term "cytokine" (e.g., GM-CSF, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-15, IL-18, IL-21, IFN-α, IFN-β, IFN-γ, MIP-1α, MIP-1β, TGF-β, TNF-α, and TNFβ) includes the full length, fragments, or variants of naturally occurring cytokines, such as functional variants (including fragments and functional variants that have at least 10%, 30%, 50%, or 80% of the activity (e.g., immunomodulatory activity) of naturally occurring cytokines). In some embodiments, the cytokine has an amino acid sequence that is substantially identical to that of a naturally occurring cytokine (e.g., at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity), or is encoded by a nucleotide sequence that is substantially identical to that of a naturally occurring nucleotide sequence encoding the cytokine (e.g., at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity). In some embodiments, as understood from the context, the cytokine also includes a receptor domain, such as a cytokine receptor domain (e.g., IL-15 / IL-15R).

[0230] The terms “effective amount” or “therapeutic effective amount” are used interchangeably herein and refer to the amount of a compound, formulation, material or composition that is effective in achieving a particular biological outcome as described herein.

[0231] As used in this article, the term "therapeutic" refers to treatment. Therapeutic effects are achieved by reducing, suppressing, alleviating, or eradicating a disease state (e.g., cancer).

[0232] As used in this article, the term “prevention” refers to preventive or protective treatment of a disease or disease state (e.g., cancer).

[0233] As used herein, "refractory" refers to a disease that does not respond to treatment, such as cancer. In one implementation, refractory cancer may be resistant to treatment prior to treatment or at the start of treatment. In other implementations, refractory cancer may become resistant during treatment. Refractory cancer is also known as drug-resistant cancer.

[0234] As used herein, “relapsed” or “recurrence” refers to the recovery or recurrence of a disease (e.g., cancer) or the signs and symptoms of a disease such as cancer after an improvement or response period, such as after prior treatment with a therapy (e.g., cancer therapy). An initial response period may include a decrease in cancer cell levels below a certain threshold, such as 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1%. Recurrence may include an increase in cancer cell levels above a certain threshold, such as 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1%. For example, in some cancer cases, recurrence may include, for example, the recurrence of a tumor after a response. In some embodiments, a response (e.g., a complete response or a partial response) may include the absence of detectable tumor or detectable MRD (minimal residual disease). In some embodiments, the initial response period lasts for at least 1, 2, 3, 4, 5, or 6 days; at least 1, 2, 3, or 4 weeks; at least 1, 2, 3, 4, 6, 8, 10, or 12 months; or at least 1, 2, 3, 4, or 5 years.

[0235] Scope: Throughout this disclosure, various embodiments of the invention may be presented in a scope format. It should be understood that the scope format is for convenience and brevity only and should not be construed as a rigid limitation on the scope of this disclosure. Accordingly, a scope description should be considered as having specifically disclosed all possible sub-scopes and the individual values ​​within those scopes. For example, a scope such as 1 to 6 should be considered as having specifically disclosed sub-scopes such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and the individual numbers within those scopes, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. As another example, a scope such as 95-99% identity includes things having 95%, 96%, 97%, 98%, or 99% identity, and includes sub-scopes such as 96-99%, 96-98%, 96-97%, 97-99%, 97-98%, and 98-99% identity. This applies regardless of the breadth of the scope.

[0236] As used herein, "adjuvant" refers to additional cancer treatment given after initial treatment to reduce the risk of cancer recurrence in some implementations of cancer treatment. Adjuvant therapies may include, for example, chemotherapy, radiation therapy, hormone therapy, targeted therapy, or biological therapy.

[0237] As used herein, "adjuvant" refers to a substance that can facilitate or amplify a cascade of immune events in some embodiments involving vaccine delivery, ultimately leading to an increased immune response, such as a comprehensive bodily response to an antigen, including cellular and / or humoral immune responses. Non-limiting examples of adjuvants include: aluminum (e.g., aluminum gels and / or aluminum salts, such as aluminum hydroxide, aluminum phosphate, and potassium aluminum sulfate), lipids (e.g., squalene, monophospholipid A (MPL)), ASO3 (e.g., an adjuvant containing D,L-α-tocopherol (vitamin E), squalene, and polysorbate 80), ASO4 (e.g., an adjuvant containing a combination of aluminum hydroxide and MPL), and MF59® (e.g., an adjuvant containing squalene).

[0238] As used herein, the term "backing" refers to a material suitable for bonding to and / or adhering to a component of a microneedle. In some embodiments, the backing material is suitable for bonding to and / or adhering to a soluble substrate of the microneedles described herein.

[0239] As used herein, the term "substrate" refers to the layer that forms the base of the microneedles (e.g., used as a support for the distal filament tips loaded with anticancer agents, immunomodulators, or combinations thereof), and / or may also be used as a layer connecting adjacent microneedles to form a continuous microneedle array or microneedle patch. In some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the substrate dissolves upon application to biological barriers such as skin, tumors, tissues, cell membranes, mucous surfaces, oral cavity, or buccal cavity.

[0240] As used herein, the term “dosage” refers to the amount of anticancer agent and / or immunomodulatory agent administered (e.g., in the microneedles described herein) to elicit an anticancer response and / or immune response (e.g., humoral and / or cellular immune response) in a living organism.

[0241] As used herein, “standard dose” refers to the amount of an anticancer agent and / or immunomodulatory agent administered at a typical human dose, such as those approved for marketing by national or international regulatory agencies (e.g., the U.S. FDA, EMEA).

[0242] As used herein, a "partial dose" refers to a dose comprising a portion of the total dose (e.g., a standard dose) of an anticancer agent and / or immunomodulatory agent, which is administered (e.g., in a microneedle) to elicit an anticancer response and / or immune response in a organism. In some embodiments, the amount of anticancer agent and / or immunomodulatory agent administered in a partial dose does not exceed 1 / X, where X is any number, e.g., where X is 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 or more times the total dose (e.g., a standard dose) of the anticancer agent and / or immunomodulatory agent administered.

[0243] As used herein, the term "gelatin" refers to a water-soluble protein derived from collagen. In some embodiments, the term "gelatin" refers to a sterile, pyrogen-free protein formulation (e.g., fraction) produced by partial acid hydrolysis (type A gelatin) or partial alkaline hydrolysis (type B gelatin) of animal collagen, most commonly derived from bovine, swine, and fish sources. Gelatin in various molecular weight ranges is available. Recombinant gelatin may also be used.

[0244] As used herein, the term "polyethylene glycol (PEG)" refers to an oligomer or polymer of ethylene oxide. PEG is also known as polyethylene oxide (PEO) or polyoxyethylene (POE). The structure of PEG is typically represented as H-(O-CH2-CH2). n -OH.

[0245] As may be used interchangeably herein, the term "sustained-release silk fibroin tip" refers to the distal end, e.g., tip, of a microneedle capable of penetrating a subject's biological barriers, such as skin, mucous surfaces, tumors, oral cavity, or buccal cavity, and depositing within the biological barrier or skin layer (e.g., dermis). In embodiments, the tip contains silk fibroin in an amount sufficient to sustain the release of therapeutic agents such as anticancer agents and / or immunomodulators for a prolonged period, e.g., at least about 1 day (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days or more, e.g., about 4 days to about 30 days, e.g., about 1-2 weeks, about 1-3 weeks, or about 1-4 weeks, e.g., about 2-12 months).

[0246] As used herein, the term "microneedle" refers to a structure having at least two, more typically three components (e.g., layers) for transporting or delivering therapeutic agents, such as anticancer agents, immunomodulators, or combinations thereof, across biological barriers such as skin, tissue, tumor, or cell membranes. In some embodiments, a microneedle comprises a base (e.g., a soluble base as described herein), a tip (e.g., an implantable tip as described herein), and optionally a backing material. In the implementation, the microneedles have a height of about 350µm to about 1500µm (e.g., about 350µm to about 1500µm, such as about 350µm, about 400µm, about 450µm, about 500µm, about 550µm, about 600µm, about 650µm, about 700µm, about 750µm, about 800µm, about 850µm, about 900µm, about 950µm, about 1000µm, about 1050µm, about 1100µm, about 1150µm, about 1200µm, about 1250µm, about 1300µm, about 1350µm, about 1400µm, about 1450µm, about 1500µm). In some embodiments, microneedles are manufactured to have any size and / or geometry that enables the deployment of silk fibroin tips (e.g., implantable, continuously releasing tips) into the dermis of the skin at a depth of about 100 μm to about 900 μm (e.g., at a depth of about 800 μm) to release (e.g., controlled or continuously released) anticancer agents, immunomodulators, or combinations thereof.

[0247] As used herein, the terms "microneedle patch" and "microneedle array" refer to a device comprising, for example, multiple microneedles (e.g., silk fibroin-based microneedles) arranged in a random or predetermined pattern such as an array. In some embodiments, the microneedle patch or microneedle array of this disclosure may contain silk fibroin in an amount from about 0.5 μg to about 500 μg. In some embodiments, the microneedle patch or microneedle array of this disclosure may be about 0.5 µg to about 5 µg, or about 1 µg to about 10 µg, or about 5 µg to about 15 µg, or about 10 µg to about 20 µg, or about 15 µg to about 25 µg, or about 20 µg to about 30 µg, or about 25 µg to about 35 µg, or about 30 µg to about 40 µg, or about 35 µg to about 45 µg, or about 40 µg to about 50 µg, or about 45 µg to about 55 µg, or about 50 µg to about 60 µg, or about 55 µg to about 65 µg, or about 60 µg to about 70 µg, or about 65 µg to about 70 µg. The amount of silk fibroin is 5µg, or about 70µg to about 80µg, or about 75µg to about 85µg, or about 80µg to about 90µg, or about 85µg to about 95µg, or about 90µg to about 100µg, or about 95µg to about 150µg, or about 125µg to about 175µg, or about 150µg to about 200µg, or about 225µg to about 275µg, or about 250µg to about 300µg, or about 325µg to about 375µg, or about 350µg to about 400µg, or about 425µg to about 475µg, or about 450µg to about 500µg. In some embodiments, the microneedle patch or microneedle array of this disclosure may contain at least about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450 or 500 µg of silk fibroin. In some embodiments, the microneedle patch or microneedle array of this disclosure may contain up to about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450 or 500 µg of silk fibroin.In some embodiments, the microneedle patch or microneedle array of this disclosure may contain an amount of silk fibroin of about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450 or 500 µg.

[0248] In some embodiments, the microneedle patch or microneedle array of this disclosure comprises about 1% to about 75%, about 1% to about 5%, about 10% to about 60%, about 15% to about 50%, or about 20% to about 40% of silk fibroin by weight. In some embodiments, the microneedle patch or microneedle array of this disclosure comprises at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75% of silk fibroin by weight. In some embodiments, the microneedle patch or microneedle array of this disclosure comprises at most about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75% of silk fibroin by weight. In some embodiments, the microneedle patch or microneedle array of this disclosure contains about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75% silk fibroin by weight.

[0249] As used herein, the term "silk fibroin" includes both silkworm silk fibroin and insect or spider silk fibroin. Any type of silk fibroin may be used depending on the aspects described herein. From silkworms such as the domestic silkworm (… Bombyx mori Silk fibroin, produced from silkworm cocoons, is the most common and earth-friendly renewable resource. For example, silk fibroin used in microneedles (e.g., silk fibroin tips, such as implantable controlled or sustained-release tips of microneedles) can be obtained by removing sericin from silkworm cocoons. In some embodiments, the silk fibroin is regenerated silk fibroin, such as silk fibroin obtained after extracting sericin from silkworm cocoons and undergoing additional treatment, such as a boiling step. Organic silkworm cocoons are also commercially available. However, many different types of silk exist, including spider silk (e.g., from golden silkworms...). Nephila clavipes(obtained), transgenic silk, recombinant and / or genetically engineered silk, such as silk derived from bacteria, yeast, mammalian cells, transgenic animals or transgenic plants (see, for example, WO 97 / 08315; US5,245,012) and its variants.

[0250] As used herein, “subject” refers to a human or animal. Typically, an animal is a vertebrate such as a primate, rodent, domesticated animal, or game animal. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques (e.g., rhesus monkeys). Rodents include mice, rats, marmots, ferrets, rabbits, and hamsters. Domesticated and game animals include cattle, horses, pigs, deer, bison, buffalo, felines (e.g., domestic cats), canines (e.g., dogs, foxes, wolves), birds (e.g., chickens, emus, ostriches), and fish (e.g., trout, catfish, and salmon). In some embodiments of the aspects described herein, the subject is a mammal (e.g., a primate, such as a human). The subject can be male or female. In some embodiments, the subject is a mammal. Mammals can be humans, non-human primates, mice, rats, dogs, cats, horses, or cattle, but are not limited to these examples. Additionally, the methods and formulations described herein can be used to treat domesticated animals and / or pets. In some implementations, the term "subject" is intended to include a living organism (e.g., a mammal, such as a human) in which an immune response can be elicited.

[0251] As used herein, the terms “release” and “controlled or sustained release” refer to the release of a therapeutic agent such as an anticancer agent, an immunomodulatory agent, or a combination thereof over a period of time (e.g., from the microneedles, microneedle devices, formulations, compositions, articles, devices, or formulations described herein, e.g., from the tips of silk fibroin-based microneedles as described herein), for example, for at least about 1 day to about 28 days (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 days or more, e.g., about 4 days to about 14 days, e.g., about 1-2 weeks, about 1-3 weeks, or about 1-4 weeks, e.g., about 1 month to about 3 months). In some embodiments, the controlled or sustained release of therapeutic agents such as anticancer agents, immunomodulators, or combinations thereof from microneedles, microneedle devices, formulations, compositions, articles, devices, or formulations as described herein over a period of about 1 day to about 14 days, such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days, may, for example, induce an anticancer response and / or immune response in a subject. In some embodiments, the controlled or sustained release of therapeutic agents such as anticancer agents, immunomodulators, or combinations thereof from microneedles, microneedle devices, formulations, compositions, articles, devices, or formulations as described herein over a period of about 1 week to about 4 weeks, such as about 1, 2, 3, or 4 weeks, may, for example, induce an anticancer response and / or immune response in a subject. In some embodiments, formulations and formulations comprising silk fibroin and therapeutic agents such as anticancer agents, immunomodulators, or combinations thereof have controlled or sustained release properties (e.g., formulated and / or configured for release over time periods of 1, 5, 10, 15, 30, 45 minutes or at least 1, 5, 10, 15, 30, 45 minutes; over time periods of 1, 2, 3, 4, 5, 10, 24 hours or at least 1, 2, 3, 4, 5, 10, 24 hours; over time periods of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days or at least 1 day). The therapeutic agent may be released into the skin of a subject over time periods of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days; over time periods of 1, 2, 3, 4, 5, 6, 7, or 8 weeks or at least 1, 2, 3, 4, 5, 6, 7, or 8 weeks; over time periods of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 months or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 months; or over time periods of 1, 2, 3, 4, or 5 years or longer or at least 1, 2, 3, 4, or 5 years or longer. In some embodiments, controlled or sustained release is achieved through abrupt release. In some embodiments, the microneedles and microneedle devices described herein may be configured for sustained release of therapeutic agents such as chemotherapy agents for approximately 28 days or longer.

[0252] As used herein, the terms “therapeutic agent” and “active agent” are recognized terms in the art and refer to any chemical portion of a biologically, physiologically, or pharmacologically active substance that acts locally or systemically in a subject. Various forms of therapeutic agents may be used that can be released from the microneedles described herein into adjacent tissues or fluids after administration to a subject.

[0253] Various embodiments of the compositions and methods described herein will be described in more detail below. Additional definitions are set forth throughout the specification. Detailed Implementation

[0254] This article provides fibroin-based microneedles and fibroin-based microneedle devices (e.g., microneedle patches) configured to introduce and subsequently release (e.g., administer) an effective amount of a therapeutic agent (e.g., an anticancer agent, an immunomodulator, or a combination thereof) into a subject (e.g., release into the subject's biological barriers such as the skin and / or release across the subject's biological barriers such as the skin). The use of fibroin-based microneedles and fibroin-based microneedle devices (e.g., microneedle patches) can generate anticancer effects and / or cancer-resistant immunity in patients (e.g., prolonged broad-spectrum immunity against cancer-associated antigens (e.g., neoantigens)).

[0255] Without being bound by theory, the application of the microneedles or microneedle devices disclosed herein to a subject at an application site (e.g., a site of abnormal cells, such as a tumor or lesion) results in the release of an effective amount of a therapeutic agent (e.g., an anticancer agent, an immunomodulator, or a combination thereof) into the subject, thereby inducing a local therapeutic effect (e.g., a local anticancer effect) at or near the application site. In addition to this local therapeutic effect, the application of the microneedles or microneedle devices disclosed herein can also produce a systemic therapeutic effect (e.g., a systemic anticancer effect) at a distant site having similar characteristics to the application site (e.g., a distant site of abnormal cells, such as a distant tumor or distant lesion).

[0256] In some embodiments, the microneedles or microneedle devices disclosed herein can be administered to a subject to achieve sustained release of an effective amount of a therapeutic agent (e.g., an anticancer agent, an immunomodulator, or a combination thereof) into the subject over a predetermined time period (e.g., 4-15 days). In some embodiments, the microneedles or microneedle devices disclosed herein can be administered to a subject to achieve sustained release of an effective amount of a therapeutic agent (e.g., an anticancer agent, an immunomodulator, or a combination thereof) into the subject over a predetermined time period, such as for time periods of at least 1, 5, 10, 15, 30, or 45 minutes; for time periods of 1, 2, 3, 4, 5, 10, or 24 hours; or for time periods of at least 1, 2, 3, 4, 5, 10, or 24 hours; or for days of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days. Or within a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days; within a period of 1, 2, 3, 4, 5, 6, 7, 8 weeks or at least 1, 2, 3, 4, 5, 6, 7, 8 weeks; within a period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 months or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 months; within a period of 1, 2, 3, 4, 5 years or longer or at least 1, 2, 3, 4, 5 years or longer.

[0257] In some embodiments, the microneedles or microneedle devices disclosed herein may be administered to a subject to achieve the sudden release of an effective amount of a therapeutic agent (e.g., an anticancer agent, an immunomodulatory agent, or a combination thereof) into the subject over a predetermined time period (e.g., about 24 hours or less). In some embodiments, the microneedles or microneedle devices disclosed herein may be administered to a subject to achieve the sudden release of an effective amount of a therapeutic agent (e.g., an anticancer agent, an immunomodulatory agent, or a combination thereof) into the subject over a predetermined time period, such as over a time period of at least 1, 5, 10, 15, 30, or 45 minutes; or over a time period of 1, 2, 3, 4, 5, 10, or 24 hours, or over a time period of at least 1, 2, 3, 4, 5, 10, or 24 hours.

[0258] In some implementations, the microneedles or microneedle devices disclosed herein may be administered in combination with a second therapeutic agent or procedure.

[0259] Therefore, this article discloses a method for manufacturing an improved silk fibroin-based microneedle for treating diseases in subjects and a device comprising the microneedle.

[0260] Silk fibroin-based microneedles In some embodiments, this disclosure provides silk fibroin-based microneedles and microneedle devices (e.g., microneedle patches) for delivering and releasing effective amounts of therapeutic agents such as anticancer agents, immunomodulators, or combinations thereof into and / or across biological barriers (e.g., skin, tumors, mucous membranes, tissues such as organ tissues and muscle tissues, buccal cavity, oral cavity, or cell membranes).

[0261] Therefore, the silk fibroin-based microneedles and microneedle devices disclosed herein can be configured to have various mechanical properties (e.g., strength), designs and geometries (e.g., needle shape and sharpness) and release kinetics (e.g., sustained release and / or sudden release) to enable the administration of effective amounts of therapeutic agents such as anticancer agents, immunomodulators, or combinations thereof to a subject for, for example, to treat diseases or conditions such as cancer and / or skin conditions.

[0262] Mechanical properties Microneedles, including the silk fibroin-based microneedles disclosed herein, can be designed to be inserted into the skin without breaking. In some embodiments, microneedle insertion is achieved by using a needle with a sharp tip and sufficient length to overcome deflection of the surface of a biological barrier (e.g., skin) that occurs prior to insertion. In some embodiments, the integrity of the microneedle during insertion can be achieved by minimizing the required insertion force, for example, by using a needle with a sharp tip, by maximizing mechanical strength, and / or by optimizing the needle diameter (see, for example, Park et al., J. Korean Phys. Soc. 56(4): 1223-1227, 2010). Therefore, in some embodiments, the mechanical properties of the silk fibroin-based microneedles are optimized (e.g., by adjusting the concentrations of various formulation components, including the crystallinity of the silk fibroin disclosed herein) to avoid sudden breakage of the microneedles due to buckling and to enable the microneedles to successfully penetrate and insert into biological barriers (e.g., skin, tumors, tissues, cell membranes, mucous surfaces, oral cavity, or buccal cavity). In some embodiments, the microneedles disclosed herein may be configured to have a geometry with a length-to-equivalent diameter aspect ratio of less than 4:1 and / or mechanical strength characterized by a Young's modulus greater than 500 MPa and a breaking stress greater than 10 MPa. In some embodiments, the microneedles have an included angle of 15 degrees and / or an aspect ratio of about 4:1. In some embodiments, the substrate formulation has a flexural modulus of about 1000 to about 1500 MPa and a breaking stress of about 15 to about 30 MPa. In some embodiments, the microneedles disclosed herein may be configured to have a geometry with a length-to-equivalent diameter aspect ratio of less than 2:1. In some embodiments, the microneedles have an included angle of about 5 degrees to about 50 degrees. For example, the microneedles may have included angles of about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 degrees. In some embodiments, the microneedles have an included angle of 30 degrees. In some embodiments, the microneedles have an included angle of 30 degrees and / or an aspect ratio of about 1.87:1. In some embodiments, the substrate formulation has a flexural modulus of about 50 to about 1500 MPa (e.g., about 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 560, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400 or 1500 MPa) and / or a flexural failure stress of about 1 to about 30 MPa (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30 MPa).

[0263] microneedle design In some embodiments, this disclosure provides fibroin-based microneedles with various design configurations and devices comprising them. The fibroin-based microneedles disclosed herein can be of any shape and / or geometry suitable for penetrating biological barriers (e.g., skin, tumors, tissues, cell membranes, mucous surfaces, oral cavity, or buccal cavity) to enable the release of therapeutic agents (e.g., anticancer agents, immunomodulators, or combinations thereof) within the subject, such as sustained release and / or sudden release. Non-limiting examples of the shape and / or geometry of the microneedles include: cylindrical shapes, wedge shapes, conical shapes, pyramidal shapes, and / or irregular shapes, or any combination thereof.

[0264] In some embodiments, the silk fibroin-based microneedles comprise soluble and / or degradable microneedles. In some embodiments, the soluble and / or degradable (e.g., reabsorbable) microneedles of this disclosure encapsulate therapeutic agents (e.g., anticancer agents, immunomodulators, or combinations thereof) in a formulation such as a silk fibroin-based formulation, which dissolves and / or degrades once within a subject (e.g., skin). In some embodiments, the release of the therapeutic agent (e.g., anticancer agents, immunomodulators, or combinations thereof) from the degradable microneedles is via protease-mediated degradation. In some embodiments, only a portion of the silk fibroin-based microneedles (e.g., the tip, such as a silk fibroin tip) is configured to be soluble and / or degradable. In some embodiments, substantially all of the silk fibroin-based microneedles are soluble and / or degradable. In some embodiments, the release of the therapeutic agent (e.g., anticancer agents, immunomodulators, or combinations thereof) from the soluble and / or degradable (e.g., reabsorbable) microneedles is via diffusion-controlled release through the microneedle material.

[0265] In some embodiments, the silk fibroin-based microneedles are solid microneedles. In some embodiments, the solid microneedles of this disclosure are designed as a two-part system. In some embodiments, a microneedle device comprising silk fibroin-based solid microneedles is first applied to the skin to create micropores just deep enough to penetrate the outermost layer of a biological barrier (e.g., skin), and then a therapeutic agent (e.g., an anticancer agent, an immunomodulator, or a combination thereof) is applied via a transdermal patch. In some embodiments, the release of the therapeutic agent (e.g., an anticancer agent, an immunomodulator, or a combination thereof) from the solid microneedles is achieved through diffusion through the microneedle material and / or degradation of the microneedle material, such as protease-mediated degradation. In some embodiments where the solid microneedles degrade, the solid microneedles are generally referred to as soluble or reabsorbable microneedles.

[0266] In some embodiments, the silk fibroin-based microneedles are hollow microneedles. In some embodiments, the hollow microneedles of this disclosure include a reservoir that delivers a therapeutic agent (e.g., an anticancer agent, an immunomodulator, or a combination thereof) directly to the application site (e.g., a biological barrier, such as the skin).

[0267] In some embodiments, the silk fibroin-based microneedles are coated microneedles. In some embodiments, a therapeutic agent (e.g., an anticancer agent, an immunomodulator, or a combination thereof) is applied directly to a portion (e.g., the surface) of the microneedle. In some embodiments, the coated microneedles are also coated with surfactants (e.g., octylphenol ethoxylate (e.g., Triton-X), polysorbate, poloxamer such as P188, and / or polyethoxylated alcohols) and / or thickeners to ensure proper delivery of the therapeutic agent.

[0268] In some embodiments, the silk fibroin-based microneedles of this disclosure may comprise the following layers: (1) a backing material (optional); (2) a substrate (e.g., a soluble substrate); and (3) a silk fibroin tip. For example, the microneedles described herein may comprise a backing material (optional) applied to a soluble substrate layer supporting a distal silk fibroin tip comprising silk fibroin and a therapeutic agent (e.g., an anticancer agent, an immunomodulator, or a combination thereof).

[0269] In some embodiments, the silk fibroin-based microneedles of this disclosure may contain silk fibroin in an amount from about 0.5 μg to about 500 μg. In some embodiments, the silk fibroin-based microneedles of this disclosure may contain about 0.5 µg to about 5 µg, or about 1 µg to about 10 µg, or about 5 µg to about 15 µg, or about 10 µg to about 20 µg, or about 15 µg to about 25 µg, or about 20 µg to about 30 µg, or about 25 µg to about 35 µg, or about 30 µg to about 40 µg, or about 35 µg to about 45 µg, or about 40 µg to about 50 µg, or about 45 µg to about 55 µg, or about 50 µg to about 60 µg, or about 55 µg to about 65 µg, or about 60 µg to about 70 µg, or about 65 µg to about 70 µg. The amount of silk fibroin is 5µg, or about 70µg to about 80µg, or about 75µg to about 85µg, or about 80µg to about 90µg, or about 85µg to about 95µg, or about 90µg to about 100µg, or about 95µg to about 150µg, or about 125µg to about 175µg, or about 150µg to about 200µg, or about 225µg to about 275µg, or about 250µg to about 300µg, or about 325µg to about 375µg, or about 350µg to about 400µg, or about 425µg to about 475µg, or about 450µg to about 500µg. In some embodiments, the silk fibroin-based microneedles of this disclosure may contain at least about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, or 500 µg of silk fibroin. In some embodiments, the silk fibroin-based microneedles of this disclosure may contain silk fibroin in amounts of up to about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, or 500 µg. In some embodiments, the silk fibroin-based microneedles of this disclosure may contain an amount of silk fibroin of about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, or 500 µg.

[0270] In some embodiments, the silk fibroin-based microneedles of this disclosure comprise about 1% to about 75%, about 1% to about 5%, about 10% to about 60%, about 15% to about 50%, or about 20% to about 40% of silk fibroin by weight. In some embodiments, the silk fibroin-based microneedles of this disclosure comprise at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75% of silk fibroin by weight. In some embodiments, the silk fibroin-based microneedles of this disclosure comprise at most about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75% of silk fibroin by weight. In some embodiments, the silk fibroin-based microneedles of this disclosure contain about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75% of silk fibroin by weight.

[0271] backing Exemplary backing materials that can be used to fabricate the microneedles of this disclosure include, but are not limited to, solid supports, such as paper-based materials, plastic materials, polymeric materials, or polyester-based materials (e.g., Whatman 903 paper, polymeric tape, plastic tape, adhesive-backed polyester tape, or other suitable tapes). In some embodiments, the backing includes Whatman 903 paper. In some embodiments, the backing includes polyester tape. In some embodiments, the polyester tape includes adhesive-backed polyester tape. In some embodiments, the backing material may be coated (e.g., at least on one side) with an adhesive suitable for bonding and / or adhering to a soluble substrate of the microneedles described herein. In some embodiments, the backing may extend beyond the microneedle array. In some embodiments, the adhesive coated on the backing may be adapted to adhere to the subject's skin to hold the array in place.

[0272] The backing material used in the microneedles of this disclosure may have various properties, including but not limited to the ability to bond and / or adhere to a soluble base layer to allow for release. The backing material may be strong enough to maintain the integrity of the patch, for example, if the soluble base layer has cracks or discontinuities. The backing material may be flexible enough to conform to, for example, non-flat surfaces, such as skin surfaces. In particular, the backing may be flexible enough during wear, such as after the patch is applied (e.g., pressed) into the skin. The backing may contain and / or be composed of non-soluble materials, such that the backing maintains its integrity after the patch is applied to the skin surface and during removal of the patch from the skin surface.

[0273] In some embodiments, the backing layer comprises an adhesive-coated plastic tape, a porous material, and / or an adhesive. In some embodiments, the adhesive is selected from acrylic, acrylate, cyanoacrylate, silicone, polyurethane, and synthetic rubber. In some embodiments, the adhesive comprises a material that can be cured by light exposure.

[0274] The backing can have any dimension suitable for application to a target biological barrier (e.g., skin surface). In some embodiments, the backing shape includes a circle. In some embodiments, the backing shape includes a rectangle (e.g., a square or rectangular strip). The backing may have rounded corners (e.g., a square or rectangle with rounded corners). The backing may also include, for example, an extension intended to be used as a "handle" (see example...). Figure 3 ).

[0275] The backing can have any suitable size, for example, to accommodate the microneedle array and / or better fit the intended application site. For example, the backing can have a diameter of about 5 mm to about 50 mm, such as about 6 mm to about 40 mm, about 8 mm to about 35 mm, about 10 mm to about 30 mm, about 11 mm to about 25 mm, about 12 mm to about 24 mm, about 10 mm to about 20 mm, or about 10 mm to about 15 mm. In some embodiments, the backing has a diameter of about 5 mm or greater, such as about 6 mm, 7 mm, 8 mm, 9 mm, 10 mm, 11 mm, 12 mm, 13 mm, 14 mm, 15 mm, 16 mm, 17 mm, 18 mm, 19 mm, 20 mm, 21 mm, 22 mm, 23 mm, 24 mm, 25 mm or greater.

[0276] In some embodiments, the backing is circular with a diameter of about 8 mm to about 16 mm, for example, about 8 mm, 9 mm, 10 mm, 11 mm, 12 mm, 13 mm, 14 mm, 15 mm, or 16 mm. In some embodiments, the backing is square with dimensions of about 8 mm x 8 mm, about 9 mm x 9 mm, about 10 mm x 10 mm, about 11 mm x 11 mm, about 12 mm x 12 mm, about 13 mm x 13 mm, about 14 mm x 14 mm, about 15 mm x 15 mm, or about 16 mm x 16 mm. In some embodiments, the backing is rectangular (e.g., a rectangular strip) having a width of about 8 mm or greater (e.g., about 9 mm, about 10 mm, about 11 mm, about 12 mm, about 13 mm or greater) and a length greater than the width, for example, a length of about 10 mm or greater (e.g., about 11 mm, about 12 mm, about 13 mm, about 14 mm, about 15 mm, about 16 mm, about 17 mm, about 18 mm, about 19 mm, about 20 mm, about 21 mm, about 22 mm, about 23 mm, about 24 mm, about 25 mm or greater).

[0277] In some embodiments, the backing material may be a circle with a diameter of 12 mm. In some embodiments, the backing material may be a strip 12 mm wide with a "handle" portion extending up to 12 mm beyond the edge of the 12 mm x 12 mm patch (see example). Figure 3 In some implementations, 12mm square polyester tape with an extended "handle" of approximately 12mm square can be used (see example). Figure 3 In some embodiments, the backing may be larger, for example, about 25 mm square, optionally with rounded corners. In some embodiments, the backing may be a circle with a diameter of about 25 mm. In some embodiments, the area of ​​the backing extending beyond the array can be used to hold the patch on the skin with a biocompatible skin adhesive.

[0278] Soluble substrate A basal layer (e.g., a soluble basal layer) forms the base of the needle (e.g., serving as a support for the distal silk fibroin tip on which a therapeutic agent (e.g., an anticancer agent, an immunomodulator, or a combination thereof) can be loaded). A basal layer (e.g., a soluble basal layer) can also serve as a layer connecting adjacent microneedles to form a microneedle array or microneedle patch.

[0279] In some embodiments, the basal layer (e.g., a soluble basal layer) comprises a material that can dissolve into the subject, for example, within a predetermined wearing time (e.g., about five minutes). In some embodiments, at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the soluble basal layer dissolves within a predetermined wearing time (e.g., about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, or about 10 minutes or longer) after being applied to the subject's biological barrier (e.g., skin).

[0280] The materials used in the manufacture of the soluble base layer are strong enough to allow the microneedles to penetrate the skin, and tough enough (e.g., not excessively brittle) to also allow the microneedles to be demolded during manufacturing. The soluble base layer material can withstand routine handling without catastrophic failure and retains its mechanical properties between demolding and application (e.g., it is not so hygroscopic as to dissipate due to ambient humidity). The soluble base layer material can be non-toxic and non-reactive at the dosage used in the patch. In some embodiments, the soluble base layer comprises a water-soluble component.

[0281] Non-limiting examples of materials that can be used to manufacture a base layer (e.g., a soluble base layer) include polysaccharides, disaccharides, polymers, proteins, plasticizers, and / or surfactants. In some embodiments, the base layer (e.g., a soluble base layer) comprises one or more (e.g., two or more, three or more, four or more, five or more, or all) of the following: polysaccharides (e.g., dextran); disaccharides (e.g., sucrose, maltose, and trehalose); polymers (e.g., methylcellulose, polyethylene glycol (PEG), carboxymethyl cellulose (CMC), polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), hyaluronic acid); proteins (e.g., gelatin, silk fibroin); plasticizers (e.g., glycerol, propylene glycol); and surfactants (e.g., octylphenol ethoxylates (e.g., Triton-X), polysorbates, poloxamer, and / or polyethoxylated alcohols).

[0282] The substrate layer disclosed herein may contain polysaccharides, disaccharides, polymers, proteins, plasticizers, and / or surfactants at a concentration of about 0.001% to about 75% (e.g., about 0.001% to about 1%, e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about 30%, or about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, or about 75%). In some cases, the dried solid substrate may contain polysaccharides, disaccharides, polymers, proteins, plasticizers, and / or surfactants at a concentration of up to about 100%. In some cases, the dried solid substrate may contain surfactants at a concentration of about 0.001%.

[0283] In some embodiments, the base layer (e.g., a soluble base layer) is configured for the sudden and / or sustained release of a therapeutic agent (e.g., an anticancer agent, an immunomodulator, or a combination thereof). In some embodiments, the base layer (e.g., a soluble base layer) comprises one or more of the following (e.g., two or more, three or more, four or more, five or more, or six or more): gelatin, dextran, glycerin, polyethylene glycol (PEG), sucrose, trehalose, maltose, carboxymethyl cellulose (CMC), polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), hyaluronic acid salt, methylcellulose, and / or surfactants (e.g., octylphenol ethoxylate (e.g., Triton-X), polysorbate, poloxamer, and / or polyethoxylated alcohols), optionally wherein the microneedles are configured for sustained and / or sudden release.

[0284] In some embodiments, the base layer (e.g., a soluble base layer) comprises one or more of the following (e.g., two or more, three or more, or four or more): dextran, sucrose, glycerol, and surfactants (e.g., octylphenol ethoxylate (e.g., Triton-X), polysorbate, poloxamer, and / or polyethoxylated alcohols), optionally configured for sustained release.

[0285] In some implementations, the base layer (e.g., a soluble base layer) comprises polyvinyl alcohol (PVA) and sucrose, optionally configured for sudden release.

[0286] In some embodiments, the base layer (e.g., a soluble base layer) comprises dextran. In some embodiments, the dextran may have a molecular weight of about 30 kDa to about 600 kDa. In some embodiments, the dextran has a molecular weight of about 40 kDa, about 50 kDa, about 60 kDa, about 70 kDa, about 80 kDa, about 90 kDa, about 100 kDa, about 200 kDa, about 300 kDa, about 400 kDa, about 500 kDa, or about 600 kDa. In some embodiments, a mixture of different dextrans may be used, such as a mixture of dextrans having various molecular weights. In some embodiments, the dextran may be obtained from and / or derived from a variety of bacterial sources, including but not limited to… Leuconostoc mesenteroides .

[0287] In some embodiments, the base layer (e.g., a soluble base layer) does not contain poly(acrylic acid) (PAA). In some embodiments, the soluble base layer as described herein has improved biocompatibility, for example, compared to a soluble base layer containing poly(acrylic acid) (PAA). In some embodiments, the soluble base layer material induces a reduced inflammatory response and / or reduced tissue necrosis. In some embodiments, the soluble base layer material is not a PAA and induces a reduced inflammatory response and / or reduced tissue necrosis compared to a PAA. In some embodiments, the soluble base layer material has a pH similar to that of the biological barrier in which it will dissolve, for example, a pH from about 4.0 to about 8.0, such as about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, about 6.5, about 7.0, about 7.5, or about 8.0.

[0288] In other embodiments, the basal layer comprises silk fibroin and / or a therapeutic agent. The basal layer (e.g., a soluble basal layer) may contain less than 98% (e.g., less than about 98%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1%) of the total amount (e.g., dose) of the therapeutic agent loaded into the microneedles and / or microneedle device.

[0289] In some embodiments, the base layer does not contain, for example, detectable amounts of silk fibroin and / or therapeutic agents. In some embodiments, the base layer is formulated to limit and / or reduce the amount of therapeutic agent leaking (e.g., diffusing) from the silk fibroin tip into the base layer, for example, compared to base layer formulations known in the art (e.g., base layer formulations containing PAA). In some implementations, limited and / or reduced amounts of therapeutic agent leakage (e.g., diffusion) from the silk fibroin tip can be measured, for example, as compared to a base layer formulation containing PAA, after manufacturing and storage (e.g., at about 4°C (e.g., refrigerated), about 25°C (e.g., room temperature), about 37°C (e.g., body temperature), about 45°C and / or at about 50°C); about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 5 days, or about 6 days; about 1 week, about 2 weeks, or about 3 weeks; about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, or about 11 months; or about 1 year or longer.

[0290] In some embodiments, the soluble matrix comprises about 10% to about 70% gelatin (e.g., hydrolyzed gelatin) (e.g., about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, or about 70% gelatin).

[0291] In some embodiments, the soluble matrix contains about 10% to about 70% of a plasticizer such as glycerol (e.g., about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, or about 70% glycerol). In some embodiments, the plasticizer is added to reduce brittleness. For example, the brittleness of a soluble base layer containing a plasticizer can be reduced by about 1%, about 2%, about 4%, about 6%, about 8%, about 10%, about 15%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 125%, about 150%, about 200%, about 300%, or more compared to a substantially plasticizer-free soluble base layer.

[0292] In some embodiments, the soluble substrate comprises about 0.001% to about 5% of the surfactant described herein, such as polysorbate (e.g., about 0.001% to about 1%, or about 1% to about 5% of the surfactant). In some embodiments, the surfactant is added to aid processing. In some embodiments, the surfactant is added as a plasticizer.

[0293] In some embodiments, the soluble substrate comprises about 1% to about 70% of polyethylene glycol (PEG) (e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, or about 70% PEG).

[0294] In some embodiments, the soluble base contains about 1% to about 35% sucrose (e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about 30%, or about 35% sucrose).

[0295] In some embodiments, the soluble substrate contains about 1% to about 35% CMC (e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about 30%, or about 35% CMC).

[0296] In some embodiments, the soluble matrix contains about 10% to about 70% PVP (e.g., about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, or about 70% PVP).

[0297] In some embodiments, the soluble substrate contains about 1% to about 35% PVA (e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about 30%, or about 35% PVA).

[0298] In some embodiments, the soluble substrate comprises about 1% to about 75% of hyaluronic acid salt (e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about 30%, or about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, or about 75% hyaluronic acid salt).

[0299] In some embodiments, the soluble base contains about 1% to about 75% maltose (e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about 30%, or about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, or about 75% maltose).

[0300] In some embodiments, the soluble substrate comprises about 1% to about 75% methylcellulose (e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about 30%, or about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, or about 75% methylcellulose).

[0301] In some embodiments, the soluble base layer may comprise 40% w / v hydrolyzed gelatin and 10% w / v sucrose in deionized (DI) water. Optionally, the base layer may comprise 1% low-viscosity carboxymethyl cellulose (CMC), which reduces brittleness. In some embodiments, the soluble base layer may comprise up to 50% w / v 10kD MW polyvinylpyrrolidone (PVP) in DI water; up to 20% 87% hydrolyzed 13kD MW polyvinyl alcohol (PVA) in DI water; or up to 10% CMC in DI water. The following combinations may also be suitable for manufacturing the soluble base layer: 30% PVP and 10% PVA; 37% PVP, 5% PVA and 15% sucrose; or various other proportions of PVP, PVA and sucrose.

[0302] The soluble substrate can be of any suitable shape, size, or form. For example, the soluble substrate can have a shape, size, or form suitable for accommodating a microneedle array and / or better yet, for the intended application site. In some embodiments, the shape of the soluble substrate includes a circle. In some embodiments, the shape of the soluble substrate includes a rectangle (e.g., a square or rectangular strip). The soluble substrate can have rounded corners (e.g., a square or rectangle with rounded corners) or sharp corners (e.g., a square or rectangle with sharp corners).

[0303] In some embodiments, the soluble substrate layer has a diameter of about 5 mm to about 50 mm, for example, about 6 mm to about 40 mm, about 8 mm to about 35 mm, about 10 mm to about 30 mm, about 11 mm to about 25 mm, about 12 mm to about 24 mm, about 10 mm to about 20 mm, or about 10 mm to about 15 mm. In some embodiments, the soluble substrate layer has a diameter of about 5 mm or greater, for example, about 6 mm, 7 mm, 8 mm, 9 mm, 10 mm, 11 mm, 12 mm, 13 mm, 14 mm, 15 mm, 16 mm, 17 mm, 18 mm, 19 mm, 20 mm, 21 mm, 22 mm, 23 mm, 24 mm, 25 mm or greater.

[0304] In some embodiments, the soluble substrate layer is circular with a diameter of about 8 mm to about 16 mm, for example, about 8 mm, 9 mm, 10 mm, 11 mm, 12 mm, 13 mm, 14 mm, 15 mm, or 16 mm. In some embodiments, the soluble substrate layer is square with dimensions of about 8 mm x 8 mm, about 9 mm x 9 mm, about 10 mm x 10 mm, about 11 mm x 11 mm, about 12 mm x 12 mm, about 13 mm x 13 mm, about 14 mm x 14 mm, about 15 mm x 15 mm, or about 16 mm x 16 mm. In some embodiments, the soluble substrate layer is rectangular (e.g., a rectangular strip) having a width of about 8 mm or greater (e.g., about 9 mm, about 10 mm, about 11 mm, about 12 mm, about 13 mm or greater) and a length greater than the width, for example, a length of about 10 mm or greater (e.g., about 11 mm, about 12 mm, about 13 mm, about 14 mm, about 15 mm, about 16 mm, about 17 mm, about 18 mm, about 19 mm, about 20 mm, about 21 mm, about 22 mm, about 23 mm, about 24 mm, about 25 mm or greater).

[0305] The thickness of the soluble base layer can be adjusted according to the specific microneedle device and / or the intended application of the microneedle device. In some embodiments, the thickness of the soluble base layer is between about 0.5 mm and 1 mm, for example, between about 0.55 mm and 0.95 mm, between about 0.60 mm and 0.90 mm, between about 0.65 mm and 0.85 mm, or between about 0.70 mm and 0.80 mm. In some embodiments, the thickness of the soluble base layer is about 0.50 mm or greater, for example, about 0.55 mm, about 0.60 mm, about 0.65 mm, about 0.70 mm, about 0.75 mm, about 0.80 mm, about 0.85 mm, about 0.90 mm, about 0.95 mm or greater.

[0306] A soluble base layer may comprise one or more layers, for example, using more than one base layer solution in the fabrication of a microneedle device by means of the methods described herein. In some embodiments, the soluble base layer comprises a single layer. In some embodiments, the soluble base layer comprises two layers. In some embodiments, the soluble base layer comprises three layers. In some embodiments, the soluble base layer comprises four or more layers. In some embodiments, the soluble base layer comprises more than one layer, wherein one or more of said layers are different, for example, when each base layer is formed using one or more different base layer solution compositions. In some embodiments, the soluble base layer comprises more than one layer, wherein each of said layers is different.

[0307] In some embodiments, the soluble base layer is approximately 12 mm square and 0.75 mm thick. In some embodiments, the soluble base layer may cover the entire patch (e.g., a microneedle patch). In some embodiments, the base layer may be a circle with a diameter of 12 mm or a square of 12 × 12 mm.

[0308] Silk fibroin tip The methods described herein can be used to manufacture silk fibroin tips of any shape, such as implantable, sustained-release and / or burst-release tips based on silk fibroin. Silk fibroin tips can be configured to contain and release effective amounts of therapeutic agents (e.g., anticancer agents, immunomodulators, or combinations thereof).

[0309] In some embodiments, the silk fibroin tip has a height / length of about 75 μm to about 800 μm (e.g., about 75, about 100 μm, about 125 μm, about 150 μm, about 250 μm to about 300 μm, about 300 μm to about 350 μm, about 350 μm to about 400 μm, about 400 μm to about 450 μm, about 450 μm to about 500 μm, about 500 μm to about 550 μm, about 550 μm to about 600 μm, about 600 μm to about 650 μm, about 650 μm to about 700 μm, about 700 μm to about 750 μm, about 750 μm to about 800 μm).

[0310] In some embodiments, the silk fibroin tip (e.g., an implantable tip) may have any diameter, for example, based on the type of biological barrier (e.g., skin layer, tumor, tissue, cell membrane, mucous surface, oral cavity, or buccal cavity) intended to be pierced by the tip. In some embodiments, the silk fibroin tip has a tip radius of about 10 μm or less (e.g., about 1 μm to about 10 μm, such as about 1 μm or less, about 2 μm or less, about 3 μm or less, about 4 μm or less, about 5 μm or less, about 6 μm or less, about 7 μm or less, about 8 μm or less, about 9 μm or less, or about 10 μm or less). In the implementation, the tip may have a size (e.g., diameter) of about 50 nm to about 50 μm (e.g., about 50 nm to about 250 nm, about 250 nm to about 500 nm, about 500 nm to about 750 nm, about 750 nm to about 1 μm, about 1 μm to about 5 μm, about 5 μm to about 10 μm, about 10 μm to about 15 μm, about 15 μm to about 20 μm, about 20 μm to about 25 μm, about 25 μm to about 30 μm, about 30 μm to about 35 μm, about 35 μm to about 40 μm, about 40 μm to about 45 μm, or about 45 μm to about 50 μm). It is understood that there is no fundamental limitation preventing the tip from having even smaller diameters (e.g., it has been shown that the limit for silk replica casting is a resolution of tens of nm, see, for example, Perry et al., 20 Adv. Mat. 3070 (2008)).

[0311] In some embodiments, the sharpness of the silk fibroin tip (e.g., an implantable, continuously releasing tip) is described herein as a tip radius. The molds used in the fabrication of the microneedles described herein are designed to have a tip radius of approximately 0.5 μm to approximately 10 μm (e.g., approximately 0.5 μm, 0.6 μm, 0.7 μm, 0.8 μm, 0.9 μm, 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, or 10 μm). In some embodiments, the tip radius is between approximately 20 μm and approximately 25 μm (e.g., approximately 20 μm, 21 μm, 22 μm, 23 μm, 24 μm, or 25 μm). Without being bound by theory, it can be understood that a blunter needle may require greater force to penetrate the epidermis. In embodiments, other dimensions of the silk fibroin tip (e.g., an implantable, continuously releasing tip) can be controlled by the shape of the mold and the fill volume. In some embodiments, the silk fibroin tip (e.g., an implantable, continuously releasing tip) may have an included angle of about 5 degrees to about 45 degrees (e.g., about 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40 or 45 degrees). In some embodiments, the silk fibroin tip (e.g., an implantable, continuously releasing tip) may have an included angle of about 15 to 45 degrees (e.g., about 15 degrees, about 16 degrees, about 17 degrees, about 18 degrees, about 19 degrees, about 20 degrees, about 21 degrees, about 22 degrees, about 23 degrees, about 24 degrees, about 25 degrees, about 26 degrees, about 27 degrees, about 28 degrees, about 29 degrees, about 30 degrees, about 31 degrees, about 32 degrees, about 33 degrees, about 34 degrees, about 35 degrees, about 36 degrees, about 37 degrees, about 38 degrees, about 39 degrees, about 40 degrees, about 41 degrees, about 42 degrees, about 43 degrees, about 44 degrees, or about 45 degrees).

[0312] In some embodiments, the height of the silk fibroin tip (e.g., an implantable, continuously releasing tip) can depend on the formulation and fill volume (e.g., fill volume or droplet dispensing volume), which can affect surface tension and drying kinetics. In some embodiments, the tip height can extend to half the entire height of the microneedle. In some embodiments, the height of the silk fibroin tip (e.g., an implantable, continuously releasing tip) is from about 75 μm to about 475 μm (e.g., about 75 μm, about 100 μm, about 125 μm, about 150 μm, about 175 μm, about 200 μm, about 225 μm, about 250 μm, about 275 μm, about 300 μm, about 325 μm, about 375 μm, about 400 μm, about 425 μm, or about 475 μm). In some embodiments, a portion of the tip comprises a thin “shell”-like layer approximately 5–10 μm thick (e.g., about 5, 6, 7, 8, 9, or 10 μm thick). In some implementations, the silk fibroin tip (e.g., an implantable, continuously releasing tip) can be dried into a more solid construct with a minimal “shell”, wherein the height can be closer to 150 μm (e.g., about 50 μm to about 200 μm) and the thickness >50 μm (e.g., about 25 μm to about 75 μm).

[0313] Furthermore, the microneedles of this disclosure can utilize developed techniques known in the art, such as functionalizing silk fibroin (e.g., active agents such as dyes and sensors). See, for example, U.S. Patent No. 6,287,340, Bioengineered anterior cruciate ligament; WO 2004 / 000915, Silk Biomaterials&Methods of Use Thereof; WO2004 / 001103, Silk Biomaterials&Methods of Use Thereof; WO 2004 / 062697, Silk Fibroin Materials&Use Thereof; WO 2005 / 000483, Method for Forming InorganicCoatings; WO 2005 / 012606, Concentrated Aqueous Silk Fibroin Solution&UseThereof; WO 2011 / 005381, Vortex-Induced Silk fibroin Gelation for Encapsulation&Delivery; WO 2005 / 123114, Silk-Based Drug Delivery System; WO2006 / 076711, Fibrous Protein Fusions&Uses Thereof in the Formation of AdvancedOrganic / Inorganic Composite Materials; US Application Publication No. 2007 / 0212730, CovalentlyImmobilized Protein Gradients In Three-Dimensional Porous Scaffolds; WO 2006 / 042287, Method for Producing Biomaterial Scaffolds; WO 2007 / 016524, Method for Stepwise Deposition of Silk Fibroin Coatings; WO 2008 / 085904, Biodegradable Electronic Devices; WO 2008 / 118133, Silk Microspheres for Encapsulation&Controlled Release;WO 2008 / 108838,Microfluidic Devices&Methods forFabricating Same;WO 2008 / 127404,Nanopatterned Biopolymer Device&Method ofManufacturing Same;WO 2008 / 118211,Biopolymer Photonic Crystals&Method ofManufacturing Same;WO 2008 / 127402,Biopolymer Sensor&Method of ManufacturingSame;WO 2008 / 127403,Biopolymer Optofluidic Device&Method of Manufacturing theSame;WO 2008 / 127401,Biopolymer Optical Wave Guide&Method of ManufacturingSame;WO 2008 / 140562,Biopolymer Sensor&Method of Manufacturing Same;WO 2008 / 127405,Microfluidic Device with Cylindrical Microchannel&Method forFabricating Same;WO 2008 / 106485,Tissue-Engineered Silk Organs;WO 2008 / 140562,Electroactive Biopolymer Optical&Electro-Optical Devices&Method ofManufacturing Same;WO 2008 / 150861,Method for Silk Fibroin Gelation UsingSonication;WO 2007 / 103442,Biocompatible Scaffolds&Adipose-Derived Stem Cells;WO 2009 / 155397,Edible Holographic Silk Products;WO 2009 / 100280,3-DimensionalSilk Hydroxyapatite Compositions;WO 2009 / 061823, Fabrication of Silk FibroinPhotonic Structures by Nanocontact Imprinting; WO 2009 / 126689, System & Method for Making Biomaterial Structures. ;

[0314] In various embodiments, the silk fibroin-based microneedle tip may further comprise at least one additional therapeutic agent, wherein said additional therapeutic agent may be dispersed throughout the microneedle or form at least a portion of the microneedle tip. In some embodiments, said additional therapeutic agent may be used to treat the diseases and / or conditions described herein, such as cancer. Optionally, the silk fibroin-based microneedle tip may further comprise excipients and / or adjuvants as described herein.

[0315] In some embodiments, the microneedle tip may be made of silk fibroin and may contain the therapeutic agents described herein (e.g., anticancer agents, immunomodulators, or combinations thereof). In some embodiments, because the population of professional antigen-presenting cells in the dermis is much higher than in the subcutaneous space, the tip may be designed to be deployed into the dermal layer of the skin (e.g., not into the subcutaneous space). In humans, the thickness of the dermis is approximately 1000–2000 μm (e.g., approximately 1–2 mm) depending on location and patient age and health. In rodents, the dermis is much thinner (e.g., ~100–300 μm in mice, ~800–1200 μm in rats). Not wishing to be bound by theory, using microneedles up to 650 μm high, the tip (e.g., an implantable, continuously releasing tip) may be deployed at a depth of approximately 100 μm to approximately 600 μm to achieve controlled or continuous release of the therapeutic agents as described herein.

[0316] Unbound by theory, the molecular weight of the silk fibroin solution used in the manufacture of the microneedles described herein can act as a control factor regulating the release of therapeutic agents (e.g., anticancer agents, immunomodulators, or combinations thereof) from the tip. In some embodiments, a higher molecular weight silk fibroin solution may facilitate a slower, controlled release or sustained release (e.g., reducing the initial burst release amount (e.g., the amount released on day 0) by at least about 10% and then releasing additional antigens over at least the next 4 days). In some embodiments, the controlled or sustained release of the anticancer agent, immunomodulator, or combination thereof from the tip may occur within at least about 4 days (e.g., about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days or more, e.g., about 4 days to about 15 days, e.g., about 1-2 weeks, about 1-3 weeks, or about 1-4 weeks). In some embodiments, the release occurs within about 1 week to about 2 weeks.

[0317] In embodiments, the silk fibroin solution used in the manufacture of the microneedles described herein may be a low molecular weight silk fibroin composition comprising a population of silk fibroin fragments having a defined molecular weight range, characterized in that: no more than 15% of the total number of silk fibroin fragments in the population has a molecular weight exceeding 200 kDa, and at least 50% of the total number of silk fibroin fragments in the population has a molecular weight within a specified range, wherein the specified range is about 3.5 kDa to about 120 kDa, or about 5 kDa to about 125 kDa. In other words, the silk fibroin solution used in the manufacture of the microneedles described herein may comprise a population of silk fibroin fragments having a defined molecular weight range, characterized in that: no more than 15% of the total molar number of silk fibroin fragments in the population has a molecular weight exceeding 200 kDa, and at least 50% of the total molar number of silk fibroin fragments in the population has a molecular weight within a specified range, wherein the specified range is about 3.5 kDa to about 120 kDa, or about 5 kDa to about 125 kDa (see, for example, WO2014 / 145002, which is incorporated herein by reference).

[0318] Exemplary silk fibroin (e.g., regenerated silk fibroin) solutions may have different molecular weight distributions, for example, as determined by size exclusion chromatography (SEC) (see example...). Figure 5 In some embodiments, the silk fibroin solution may be prepared, for example, according to established methods. In some embodiments, the silk fibroin solution is first boiled in 0.02M Na2CO3 to remove sericin present in the unprocessed natural silk before SEC analysis. In some embodiments, the silk fibroin composition may be prepared by boiling the silk fibroin from silkworm cocoons at atmospheric pressure and temperature. Bombyx mori The silk fibroin composition may be a composition or mixture produced by degumming silk cocoons in an aqueous sodium carbonate solution for approximately 480 minutes or less, such as less than 480 minutes, less than 400 minutes, less than 300 minutes, less than 200 minutes, less than 180 minutes, less than 120 minutes, less than 100 minutes, less than 60 minutes, less than 50 minutes, less than 40 minutes, less than 30 minutes, less than 20 minutes, less than 10 minutes, or less than a shorter time. In one embodiment, the silk fibroin composition may be a composition or mixture produced by degumming silk cocoons in an aqueous sodium carbonate solution at atmospheric pressure boiling temperature for approximately 480 minutes or less, such as less than 480 minutes, less than 400 minutes, less than 300 minutes, less than 200 minutes, less than 180 minutes, less than 120 minutes, less than 100 minutes, less than 60 minutes, less than 50 minutes, less than 40 minutes, less than 30 minutes, less than 20 minutes, less than 10 minutes, or less than a shorter time.

[0319] In some embodiments, the silk fibroin solution may be a silk fibroin solution boiled for 10 minutes (10 MB), 60 minutes (60 MB), 120 minutes (120 MB), 180 minutes (180 MB), or 480 minutes (480 MB) (see example). Figure 5 In some embodiments, therapeutic agents such as anticancer agents, immunomodulators, or combinations thereof may be formulated in a 10 MB silk fibroin solution of 1% w / v to about 10% w / v (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% w / v). In some embodiments, therapeutic agents such as anticancer agents, immunomodulators, or combinations thereof may be formulated in a 60 MB silk fibroin solution of 1% w / v to about 10% w / v (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% w / v). In some embodiments, therapeutic agents such as anticancer agents, immunomodulators, or combinations thereof may be formulated in a 120 MB silk fibroin solution of 1% w / v to about 10% w / v (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% w / v). In some embodiments, therapeutic agents such as anticancer agents, immunomodulators, or combinations thereof may be formulated in a 180MB silk fibroin solution of 1% w / v to about 10% w / v (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% w / v). In some embodiments, therapeutic agents such as anticancer agents, immunomodulators, or combinations thereof may be formulated in a 480MB silk fibroin solution of 1% w / v to about 10% w / v (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% w / v).

[0320] In some embodiments, the silk fibroin tip (e.g., an implantable continuous-release tip) may contain an amount of silk fibroin from about 0.5 μg to about 500 μg. In some embodiments, the silk fibroin tip (e.g., an implantable continuous-release tip) may contain about 0.5 µg to about 5 µg, or about 1 µg to about 10 µg, or about 5 µg to about 15 µg, or about 10 µg to about 20 µg, or about 15 µg to about 25 µg, or about 20 µg to about 30 µg, or about 25 µg to about 35 µg, or about 30 µg to about 40 µg, or about 35 µg to about 45 µg, or about 40 µg to about 50 µg, or about 45 µg to about 55 µg, or about 50 µg to about 60 µg, or about 55 µg to about 65 µg, or about 60 µg to about 70 µg, or about 65 µg... The amount of silk fibroin is from about 75 µg, or about 70 µg to about 80 µg, or about 75 µg to about 85 µg, or about 80 µg to about 90 µg, or about 85 µg to about 95 µg, or about 90 µg to about 100 µg, or about 95 µg to about 150 µg, or about 125 µg to about 175 µg, or about 150 µg to about 200 µg, or about 225 µg to about 275 µg, or about 250 µg to about 300 µg, or about 325 µg to about 375 µg, or about 350 µg to about 400 µg, or about 425 µg to about 475 µg, or about 450 µg to about 500 µg. In some embodiments, the silk fibroin tip (e.g., an implantable, continuously releasing tip) may contain at least about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, or 500 µg of silk fibroin. In some embodiments, the silk fibroin tip (e.g., an implantable, continuously releasing tip) may contain up to about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, or 500 µg of silk fibroin.In some embodiments, the silk fibroin tip (e.g., an implantable, continuously releasing tip) may contain an amount of silk fibroin of about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, or 500 µg.

[0321] In some embodiments, the silk fibroin tip (e.g., an implantable continuous-release tip) comprises about 1% to about 75%, about 1% to about 5%, about 10% to about 60%, about 15% to about 50%, or about 20% to about 40% of silk fibroin by weight. In some embodiments, the silk fibroin tip (e.g., an implantable continuous-release tip) comprises at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75% of silk fibroin by weight. In some embodiments, the silk fibroin tip (e.g., an implantable continuous-release tip) comprises at most about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75% of silk fibroin by weight. In some embodiments, the silk fibroin tip (e.g., an implantable continuous release tip) comprises about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75% of silk fibroin by weight.

[0322] Unbound by theory, the primary tunability of silk fibroin tips (e.g., implantable sustained-release tips) lies in their crystallinity, measured by β-sheet content (intermolecular and intramolecular β-sheets). This affects the solubility of the silk tip matrix and its ability to retain antigens. As the β-sheet content increases, the mechanical strength of the tip also increases. Specific therapeutic agent (e.g., anticancer agents and / or immunomodulators) release profiles are achieved by modulating the crystallinity and diffusivity of the silk matrix. This is accomplished through both the silk input material and the formulation, as well as post-treatments that increase crystallinity (e.g., water annealing, methanol / solvent annealing). In some embodiments, silk fibroin tips (e.g., implantable controlled-release or sustained-release microneedle tips) contain approximately 10% to approximately 60% (e.g., approximately 10%, approximately 20%, approximately 30%, approximately 40%, approximately 50%, approximately 60%) of β-sheet content, for example, based on a “crystallinity index,” such as the “crystallinity index” known in the art. In some implementations, silk fibroin tips (e.g., implantable controlled-release or sustained-release microneedle tips) may be formulated as particles (e.g., microparticles and / or nanoparticles).

[0323] On the other hand, this disclosure is characterized by microneedles (e.g., silk fibroin-based microneedles) that can stably contain therapeutic agents (e.g., anticancer agents, immunomodulators, or both), for example, due in part to the thermal stability properties of the microneedle composition (e.g., the thermal stability properties of the silk fibroin composition). In some embodiments, anticancer agents, immunomodulators, or both (e.g., the anticancer agents or immunomodulators described herein) are stabilized by the microneedles or microneedle devices described herein. Not wishing to be bound by theory, the ability of the microneedles or microneedle devices of this disclosure to stabilize therapeutic agents can facilitate the storage of the microneedle devices, for example, to prevent loss of the bioactivity of the therapeutic agent during storage. Furthermore, the bioactivity of the therapeutic agent can be maintained after application of the microneedles or microneedle devices. For example, the therapeutic agent can be stabilized within the silk fibroin tip, for example, to prevent loss of bioactivity during release at body temperature (e.g., controlled release) after application of the microneedles or microneedle devices.

[0324] In some embodiments, the anticancer agent and / or immunomodulator retain at least 50% (e.g., about 50%, 60%, 70%, 80%, 90%, 92%, 94%, 96%, 98%, 99%, 99.5% or more) of its original biological activity after being stored at room temperature (e.g., about 25°C) for a period of at least about 2 weeks. In some embodiments, the anticancer agent and / or immunomodulator retain at least 70%, 80%, or 90% of its original biological activity after being stored at about 25°C for a period of at least about 2 weeks (e.g., about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, or about 12 weeks). In some embodiments, the anticancer agent and / or immunomodulatory agent retains at least 60%, 70%, or 80% of its original biological activity after being stored at about 37°C for at least about 2 weeks (up to about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, or about 12 weeks). In some embodiments, the anticancer agent and / or immunomodulatory agent retains at least 50%, 60%, or 70% of its original biological activity after being stored at about 45°C for at least about 2 weeks (up to about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, or about 12 weeks).

[0325] In some embodiments, the silk fibroin-based microneedles of this disclosure will stabilize cytokines. For example, silk fibroin can stabilize cytokines (e.g., interleukins, such as IL-2) during storage at a range of temperatures (e.g., at 4°C, room temperature (e.g., 25°C), or 37°C), including long periods (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days or more, 1, 2, 3, or 4 weeks or more, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or more, or 1, 2, 3, or 4 years or more). In some embodiments, the silk fibroin-based microneedles will stabilize interleukins. In some embodiments, the silk fibroin-based microneedles will stabilize interleukin-2 (IL-2). In some embodiments, the silk fibroin-based microneedles will stabilize IL-2 for a period of at least 14 days at 4°C. In some embodiments, the silk fibroin-based microneedles stabilize IL-2 for at least 14 days at room temperature (approximately 25°C). In some embodiments, the silk fibroin-based microneedles stabilize IL-2 for at least 14 days at body temperature (approximately 37°C).

[0326] microneedle size In some embodiments, this disclosure provides silk fibroin-based microneedles having various sizes and geometries and devices comprising them.

[0327] In one implementation, the length of the silk fibroin-based microneedles can be made long enough to allow the silk fibroin tip containing a therapeutic agent (e.g., an anticancer agent, an immunomodulator, or a combination thereof) to be delivered (e.g., implanted) to a desired depth within a biological barrier (e.g., the skin) to induce an anticancer response, or to induce an immune response.

[0328] In some implementations, the biological barrier is a tumor and / or skin lesion.

[0329] In some implementations, the length of the silk fibroin-based microneedles can be from about 350 µm to about 1500 µm (e.g., about 350 µm, about 400 µm, about 450 µm, about 500 µm, about 550 µm, about 600 µm, about 650 µm, about 700 µm, about 750 µm, about 800 µm, about 850 µm, about 900 µm, about 950 µm, about 1000 µm, about 1050 µm, about 1100 µm, about 1150 µm, about 1200 µm, about 1250 µm, about 1300 µm, about 1350 µm, about 1400 µm, about 1450 µm, about 1500 µm).

[0330] In some implementations, the length of the silk fibroin-based microneedles is sufficient to enable delivery to the epidermis (e.g., approximately 10 μm to 120 μm below the skin surface).

[0331] In some implementations, the length of the silk fibroin-based microneedles is sufficient to enable delivery into the dermis (e.g., approximately 60 μm to approximately 2.1 mm below the skin surface).

[0332] In some implementations, the length of the silk fibroin-based microneedles is sufficient to enable delivery to the eye (e.g., about 10 μm to 120 μm below the surface of the eye).

[0333] In some implementations, the length of the silk fibroin-based microneedles is sufficient to enable delivery to the tumor (e.g., about 10 μm to 120 μm below the tumor surface).

[0334] In some embodiments, the microneedle is configured to implant a silk fibroin tip into the subject's biological barrier at a depth of about 100 μm to about 600 μm (e.g., the maximum penetration depth at the distal end of the tip). In some embodiments, the length of the microneedle is about 350 μm to about 1500 μm. In some embodiments, the height of the silk fibroin tip may extend to about half of the total height of the microneedle. In some embodiments, the height of the silk fibroin tip is about 75 μm to about 475 μm. In some embodiments, the silk fibroin tip includes a tip radius of about 0.5 μm to about 25 μm. In some embodiments, the silk fibroin tip includes a tip radius of about 5 μm to about 10 μm. In some embodiments, the silk fibroin tip includes an angle of about 5 degrees to about 45 degrees.

[0335] In some embodiments, the silk fibroin tip (e.g., an implantable tip) may have any diameter, for example, based on the type of biological barrier (e.g., skin layer) intended to be pierced by the tip. In some embodiments, the silk fibroin tip has a tip radius of about 10 μm or less (e.g., about 1 μm to about 10 μm, such as about 1 μm or less, about 2 μm or less, about 3 μm or less, about 4 μm or less, about 5 μm or less, about 6 μm or less, about 7 μm or less, about 8 μm or less, about 9 μm or less, or about 10 μm or less). In the implementation, the tip may have a size (e.g., diameter) of about 50 nm to about 50 μm (e.g., about 50 nm to about 250 nm, about 250 nm to about 500 nm, about 500 nm to about 750 nm, about 750 nm to about 1 μm, about 1 μm to about 5 μm, about 5 μm to about 10 μm, about 10 μm to about 15 μm, about 15 μm to about 20 μm, about 20 μm to about 25 μm, about 25 μm to about 30 μm, about 30 μm to about 35 μm, about 35 μm to about 40 μm, about 40 μm to about 45 μm, or about 45 μm to about 50 μm). It is understood that there is no fundamental limitation preventing the tip from having even smaller diameters (e.g., it has been shown that the limit for silk replica casting is a resolution of tens of nm, see, for example, Perry et al., 20 Adv. Mat. 3070 (2008)).

[0336] In some embodiments, the sharpness of the silk fibroin tip (e.g., an implantable, continuously releasing tip) is described herein as a tip radius. The molds used in the fabrication of the microneedles described herein are designed to have a tip radius of approximately 0.5 μm to approximately 10 μm (e.g., approximately 0.5 μm, 0.6 μm, 0.7 μm, 0.8 μm, 0.9 μm, 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, or 10 μm). In some embodiments, the tip radius is between approximately 20 μm and approximately 25 μm (e.g., approximately 20 μm, 21 μm, 22 μm, 23 μm, 24 μm, or 25 μm). Without being bound by theory, it can be understood that a blunter needle may require greater force to penetrate the epidermis. In embodiments, other dimensions of the silk fibroin tip (e.g., an implantable, continuously releasing tip) can be controlled by the shape of the mold and the fill volume. In some embodiments, the silk fibroin tip (e.g., an implantable, continuously releasing tip) may have an included angle of about 5 degrees to about 45 degrees (e.g., about 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, or 45 degrees). In some embodiments, the tip may have an included angle of about 15 degrees to 45 degrees (e.g., about 15 degrees, about 16 degrees, about 17 degrees, about 18 degrees, about 19 degrees, about 20 degrees, about 21 degrees, about 22 degrees, about 23 degrees, about 24 degrees, about 25 degrees, about 26 degrees, about 27 degrees, about 28 degrees, about 29 degrees, about 30 degrees, about 31 degrees, about 32 degrees, about 33 degrees, about 34 degrees, about 35 degrees, about 36 degrees, about 37 degrees, about 38 degrees, about 39 degrees, about 40 degrees, about 41 degrees, about 42 degrees, about 43 degrees, about 44 degrees, or about 45 degrees).

[0337] Without being bound by theory, those skilled in the art can adjust the length of silk fibroin-based microneedles for a variety of factors, including but not limited to tissue thickness, such as skin thickness (e.g., varying with age, sex, body location, subject species (e.g., human)), drug delivery characteristics, diffusion properties of therapeutic agents (e.g., ionic charge and / or molecular weight, and / or shape of the therapeutic agent), or any combination thereof.

[0338] However, not wanting to be bound by theory, the use of microneedles approximately 650 μm high allows the silk fibroin tip to be deployed (e.g., implanted) into the dermis of the subject's skin at a depth of approximately 100 μm to approximately 600 μm to achieve the release (e.g., sudden or sustained release) of a therapeutic agent (e.g., an anticancer agent, an immunomodulator, or a combination thereof) from the silk fibroin tip. In some embodiments, the microneedles may be approximately 800 μm high (e.g., approximately 500 μm to 1200 μm high).

[0339] Silk fibroin-based microneedle devices for combination therapy In some embodiments, the silk fibroin-based microneedle device (e.g., a microneedle patch) of this disclosure may comprise multiple microneedles of the disclosed type. Alternatively, the microneedle device may comprise multiple microneedles of different types.

[0340] For example, a silk fibroin-based microneedle device may comprise multiple microneedles containing anticancer agents. In some embodiments, the silk fibroin-based microneedle device may comprise multiple microneedles containing combinations of anticancer agents (e.g., two, three, four, five, six, or more anticancer agents). For microneedles configured to deliver combinations of anticancer agents (e.g., two, three, four, five, six, or more anticancer agents), the microneedle device may comprise, for example, multiple identical microneedles containing (e.g., all) the same combination of anticancer agents; or multiple different microneedles containing different anticancer agents or different combinations of anticancer agents. In some embodiments, multiple microneedles contain one anticancer agent. In some embodiments, multiple microneedles contain two or more anticancer agents (e.g., two, three, four, five, six, or more anticancer agents).

[0341] In some embodiments, the silk fibroin-based microneedle device may comprise multiple microneedles containing immunomodulatory agents. In some embodiments, the silk fibroin-based microneedle device may comprise multiple microneedles containing combinations of immunomodulatory agents (e.g., two, three, four, five, six, or more anticancer agents). For microneedles configured to deliver combinations of immunomodulatory agents (e.g., two, three, four, five, six, or more immunomodulatory agents), the microneedle device may comprise, for example, multiple identical microneedles containing (e.g., all) the same combination of immunomodulatory agents; or multiple different microneedles containing different immunomodulatory agents or different combinations of immunomodulatory agents. In some embodiments, multiple microneedles contain one immunomodulatory agent. In some embodiments, multiple microneedles contain two or more immunomodulatory agents (e.g., two, three, four, five, six, or more immunomodulatory agents).

[0342] In some implementations, the silk fibroin-based microneedle device may comprise multiple microneedles containing a combination of anticancer agents and immunomodulators. For microneedles configured to deliver the combination of anticancer agents and immunomodulators, the microneedle device may comprise, for example, multiple identical microneedles containing the same combination of anticancer agents and immunomodulators; or multiple different microneedles containing different combinations of anticancer agents and immunomodulators.

[0343] In some implementations, for microneedles configured to deliver a combination of anticancer agents and immunomodulators, the microneedle device may comprise: multiple microneedles containing one anticancer agent; multiple microneedles containing two or more anticancer agents (e.g., two, three, four, five, six or more anticancer agents); multiple microneedles containing one immunomodulator; multiple microneedles containing two or more immunomodulators (e.g., two, three, four, five, six or more immunomodulators); and / or multiple microneedles containing both anticancer agents (e.g., one, two, three, four, five, six or more anticancer agents) and immunomodulators (e.g., one, two, three, four, five, six or more immunomodulators).

[0344] In some embodiments, multiple microneedles can be arranged in a random or predetermined pattern to form microneedles or patches, as described herein. Patches may comprise a carrier, backing, or "handle" layer that adheres to the back of a substrate (see, for example...). Figure 3 This layer provides structural support and a zone through which the patch can be gripped and manipulated without interfering with the needle array.

[0345] The microneedle devices (e.g., microneedle patches) described herein can be designed to accommodate different numbers of microneedles. In some embodiments, the microneedle device contains at least 50 microneedles, such as at least about 60, about 64, about 70, about 80, about 81, about 90, about 100, about 110, about 121, about 144, about 150, about 169, about 175, about 196, about 200, about 225, about 250, about 256, about 289, about 300, or more microneedles. In some embodiments, the microneedle device (e.g., a microneedle patch) contains about 50 to 500 microneedles, such as about 50 to 400 microneedles, about 75 to about 300 microneedles, about 100 to 200 microneedles, or about 100 to 150 microneedles. The microneedles can be arranged in a grid, such as a square grid. In some embodiments, the microneedles are arranged in an 8×8, 9×9, 10×10, 11×11, 12×12, 13×13, 14×14, 15×15, 16×16, or 17×17 grid. In some embodiments, the microneedles are arranged in an 11×11 grid. In some embodiments, the microneedle device has a spacing of about 0.5 mm to 1.0 mm, for example, about 0.55 mm, 0.60 mm, 0.65 mm, 0.70 mm, 0.75 mm, 0.80 mm, 0.85 mm, 0.95 mm, or 1 mm. In some embodiments, the microneedle device has a spacing of about 0.75 mm.

[0346] In some embodiments, the microneedle device comprises approximately 121 needles arranged in an 11×11 square grid, spaced approximately 0.75 mm apart. In some embodiments, the microneedle device (e.g., a microneedle patch) may comprise approximately 121 needles arranged in an 11×11 square grid, spaced approximately 0.75 mm apart. In some embodiments, the microneedle device comprises individual needles, each needle being a cone approximately 0.65 mm long with a base diameter of approximately 0.35 mm and an included angle of approximately 30°. In some embodiments, the microneedle device comprises individual needles with fibroin tips, wherein the fibroin tips are sufficiently sharp to penetrate biological barriers (e.g., skin). In some embodiments, the microneedle device comprises individual needles with fibroin tips, wherein the radius of curvature of the fibroin tips ideally should not exceed 0.01 mm.

[0347] The exemplary microneedles and devices disclosed herein Figure 2-4 Described in the text.

[0348] Release kinetics The disclosed silk fibroin-based microneedles can be configured to release therapeutic agents or combinations of therapeutic agents (e.g., anticancer agents, immunomodulators, or combinations thereof) according to various release kinetics.

[0349] In some embodiments, the silk fibroin-based microneedles can be configured to release effective amounts of anticancer agents and / or immunomodulators to induce an anticancer response in a subject. In some embodiments, the release of the anticancer agents and / or immunomodulators will enhance the subject's immune system's exposure to cancer-associated neoantigens, thereby inducing specific immune effector cells, such as T cells, specific to the neoantigens to be activated and / or amplified in the subject. The release of the anticancer agents and / or immunomodulators may promote the development of prolonged immunity against cancer in the subject.

[0350] In some embodiments, the silk fibroin-based microneedles may be configured to release a therapeutic agent or combination of therapeutic agents (e.g., an anticancer agent, an immunomodulator, or a combination thereof) via a sudden release. In some embodiments, the sudden release includes the rapid administration of an anticancer agent and / or immunomodulator to a subject. The sudden release may include the rapid administration of greater than 0% to about 100% of the total amount of anticancer agent and / or immunomodulator present in the silk fibroin tip (e.g., about 1% to about 25%, about 25% to about 50%, about 50% to about 75%, about 75% to about 100%). In some embodiments, the sudden release occurs over a time period including at least about 1 hour (e.g., about 1 to about 30 minutes, such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 24 hours).

[0351] In some embodiments, silk fibroin-based microneedles may be configured to release a therapeutic agent or combination of therapeutic agents (e.g., an anticancer agent, an immunomodulator, or a combination thereof) via sustained release. Examples of sustained release include, but are not limited to, zero-order release, primary release, and secondary release. In some embodiments, zero-order release is a release rate independent of the therapeutic agent concentration in the dosage form (e.g., microneedle). In some embodiments, zero-order release is a substantially constant release of therapeutic agent over a period of time (e.g., a constant amount of therapeutic agent released per unit time). In some embodiments, primary release is a release rate that is a function of the amount of therapeutic agent remaining in the dosage form (e.g., microneedle). In some embodiments, primary release is a constant percentage, for example, of the therapeutic agent released per unit time from the dosage form (e.g., microneedle). In some embodiments, secondary release is a release rate that quadruples when the therapeutic agent concentration in the dosage form is doubled. In some embodiments, sustained release includes substantially continuous low-dose administration of an anticancer agent and / or immunomodulator. Sustained release may include approximately 0% to approximately 100% of the total amount of anticancer agent and / or immunomodulatory agent present in the silk fibroin tip (e.g., approximately 1% to approximately 25%, approximately 25% to approximately 50%, approximately 50% to approximately 75%, approximately 75% to approximately 100%). In some embodiments, sustained release is over a period of time including at least approximately 3 days (e.g., approximately 3, 4, 5, 6, 7 or more days, e.g., approximately 5 days to approximately 10 days, e.g., approximately 7 days to approximately 15 days, e.g., approximately 1 week to approximately 2 weeks, approximately 1 week to approximately 3 weeks, or approximately 2 weeks to approximately 4 weeks, e.g., approximately 1 month to approximately 3 months, e.g., approximately 2 months to approximately 4 months, e.g., approximately 3 months to approximately 6 months). In some embodiments, sustained release is over a period of approximately 7 days to approximately 15 days.

[0352] In some implementations, the release (e.g., administration) of a therapeutic agent (e.g., an anticancer agent, an immunomodulator, or a combination thereof) from the silk fibroin-based microneedles described herein can be facilitated by diffusion of the therapeutic agent from the microneedles or portions thereof.

[0353] In some implementations, the release (e.g., administration) of a therapeutic agent (e.g., an anticancer agent, an immunomodulator, or a combination thereof) from the silk fibroin-based microneedles described herein can be facilitated by the degradation (e.g., protease-mediated degradation) of the microneedles or portions thereof.

[0354] In some implementations, the release (e.g., administration) of a therapeutic agent (e.g., an anticancer agent, an immunomodulator, or a combination thereof) from the silk fibroin-based microneedles described herein can be facilitated by the dissolution of the microneedles or portions thereof.

[0355] Without being bound by theory, the release of the anticancer agent can occur at substantially the same rate as (e.g., simultaneously) the release of the immunomodulator. In other embodiments, the release of the anticancer agent can occur at a different rate than the release of the immunomodulator, such that the anticancer agent is released substantially before or substantially after the release of the immunomodulator.

[0356] Therapeutic agents In some embodiments, this disclosure provides silk fibroin-based microneedles comprising therapeutic agents (e.g., anticancer agents, immunomodulators, or combinations thereof). This disclosure also provides combination therapies for administering the microneedles disclosed herein in combination with other therapies. Non-limiting examples of therapeutic agents that may be incorporated into the silk fibroin-based microneedles of this disclosure and / or administered in combination therapies with the silk fibroin-based microneedles of this disclosure are disclosed below.

[0357] anticancer agents In some embodiments, the silk fibroin-based microneedles of this disclosure may comprise and / or be administered in combination with low molecular weight or small molecular weight chemotherapeutic agents. Exemplary low molecular weight or small molecular weight chemotherapeutic agents include, but are not limited to, 13-cis-retinoic acid (isotretinoin, ACCUTANE®), 2-CdA (2-chlorodeoxyadenosine, cladribine, LEUSTATIN™), 5-azacytidine (azacitidine, VIDAZA®), 5-fluorouracil (5-FU, fluorouracil, ADRUCIL®), 6-mercaptopurine (6-MP, mercaptopurine, PURINETHOL®), 6TG (6-thioguanine, thioguanine, THIOGUANINE TABLOID®), abraxane (albumin-bound paclitaxel), and actinomycin-D. (COSEMGEN®), Panretin®, ATRA (Vitamin A), HEXALEN®, Methotrexate (Methotrexate, Methotrexate Sodium, MTX, TREXALL™, RHEUMATREX®), Ethylamine®, Cytarabine® (Ara-C, Cytarabine, CYTOSAR-U®), Arsenic Trioxide®, Asparaginase® (Erwinia L-Asparaginase, L-Asparaginase, ELSPAR®, KIDROLASE®), BCNU (Carbendazim, BiCNU®), Bendamustine (TREANDA®), Bexarotin (TARGRETIN®), Bleomycin (BLENOXANE®), Busulex® (MYLERAN®), Calcium Tetrahydroacetate (Citralotropic Factor, Tetrahydroacetic Acid, Leucovorin), Camptosar-11 (CPT-11, Irinotecan, CAMPTOSAR®), Capecitabine (XELODA®), Carboplatin (PARAPLATIN®), Carmustine Chip (Prolifeprospan 20 with Carmustine Implant, GLIADEL® Chip), CCI-779 (Tesirolimus, TORISEL®), CCNU (Lomustine, CeeNU), CDDP (Cisplatin, PLATINOL®, PLATINOL-AQ®), Chlorobutazone (Rubin), Cyclophosphamide (CYTOXAN®, NEOSAR®), Dacarbazine (DIC, DTIC, Imidazole Carboxamide, DTIC-DOME®), Daunorubicin (Daunorubicin, Daunorubicin Hydrochloride, CERUBIDINE®), Decitabine (DACOGEN®), ZINECARD®, DHAD (Mitoxantrone,NOVANTRONE®, TAXOTERE®, ADRIAMYCIN®, RUBEX®, ELLENCE™, HALAVEN®, EMCYT®, Etoposide (VP-16, Etoposide Phosphate, TOPOSAR®, VEPESID®, ETOPOPHOS®), FUDR®, FLUDARA®, CARAC™, EFUDEX®, FLUOROPLEX® (Cream), GEMZAR®, HYDREA®, DROXIA™, MYLOCEL™, IDAMYCIN®, IFEX®, IXEMPRA™, LCR (VCR, ONCOVIN®, VINCASARPFS®), L-PAM (L-sarcomain, melphalan, phenylalanine nitrogen mustard, ALKERAN®), dichloroethyl methylamine (dichloroethyl methylamine hydrochloride, nitrogen mustard, MUSTARGEN®), sodium thiosulfate (MESNEX™), mitomycin (mitomycin-C, MTC, MUTAMYCIN®), nerabine (ARRANON®), oxaliplatin (ELOXATIN™), paclitaxel (TAXOL®, ONXAL™), pemetrexed (PEG-L-asparaginase, ONCOSPAR®), pemetrexed (ALIMTA®), pentostatin (NIPENT®), procarbazine (MATULANE®), streptozocin (ZANOSAR®), temozolomide (TEMODAR®), teniposide (VM-26, VUMON®), TESPA (Thiophosphoramide, thiotepa, TSPA, THIOPLEX®), topotecan (HYCAMTIN®), vinblastine (vinblastine sulfate / vincaleukoblastine, VLB, ALKABAN-AQ®, VELBAN®), vinorelbine (vinorelbine tartrate, Navelbine®), and vorinostat (ZOLINZA®).

[0358] In some embodiments, the silk fibroin-based microneedles of this disclosure may comprise and / or be administered in combination with FDA-approved targeted therapies. For example, for the treatment of melanoma, the silk fibroin-based microneedles of this disclosure may comprise and / or be administered in combination with bismuthinib (MEKTOVI®), cobimetinib (COTELLIC®), dabrafenib (TAFINLAR®), caninefenib (BRAFTOVI®), trametinib (MEKINIST®), and / or vemurafenib (ZELBORAF®) and / or be administered in combination with bismuthinib (MEKTOVI®), cobimetinib (COTELLIC®), dabrafenib (TAFINLAR®), caninefenib (BRAFTOVI®), trametinib (MEKINIST®), and / or vemurafenib (ZELBORAF®).

[0359] In some embodiments, for the treatment of basal cell carcinoma, the silk fibroin-based microneedles of this disclosure may comprise ODOMZO® and / or Erivedge® and / or may be administered in combination with ODOMZO® and / or Erivedge®.

[0360] In some embodiments, for the treatment of breast cancer, the silk fibroin-based microneedles of this disclosure may comprise VERZENIO®, PIQRAY®, LYNPARZA®, IBRANCE®, KISQALI®, and / or TALZENNA® and / or may be administered in combination with VERZENIO®, PIQRAY®, LYNPARZA®, IBRANCE®, KISQALI®, and / or TALZENNA®.

[0361] In some embodiments, the silk fibroin-based microneedles of this disclosure may comprise and / or be administered in combination with biological agents. Biological agents that can be used in cancer treatment are known in the art, and the silk fibroin-based microneedles described herein may be administered, for example, in combination with such known biological agents. For example, the FDA has approved the following biologics for the treatment of breast cancer: HERCEPTIN® (trastuzumab, Genentech Inc., South San Francisco, California; a humanized monoclonal antibody with antitumor activity in HER2-positive breast cancer); FASLODEX® (fulvestrant, AstraZeneca Pharmaceuticals, LP, Wilmington, Delaware; an estrogen receptor antagonist for the treatment of breast cancer); ARIMIDEX® (anastrozole, AstraZeneca Pharmaceuticals, LP; a nonsteroidal aromatase inhibitor that blocks aromatase, an enzyme required for estrogen production); AROMASIN® (exemestane, Pfizer Inc., New York City, NY; an irreversible steroidal aromatase inactivator for the treatment of breast cancer); FEMARA® (letrozole, Novartis Pharmaceuticals, East Hanover, NJ; an FDA-approved nonsteroidal aromatase inhibitor for the treatment of breast cancer); and NOLVADEX® (tamoxifen, AstraZeneca Pharmaceuticals, LP; an FDA-approved nonsteroidal anti-estrogenic agent for the treatment of breast cancer. Other biologics that can be combined with the silk fibroin-based microneedles disclosed herein include: AVASTIN® (bevacizumab, Genentech Inc.; the first FDA-approved therapy designed to inhibit angiogenesis); and ZEVALIN® (tiemomumab, Biogen Idec (Cambridge, Massachusetts; a radiolabeled monoclonal antibody currently approved for the treatment of B-cell lymphoma).

[0362] In addition, the FDA has approved the following biologics for the treatment of colorectal cancer: AVASTIN®; ERBITUX® (cetuximab, ImClone Systems Inc. (New York City, NY) and Bristol-Myers Squibb (New York City, NY); a monoclonal antibody targeting the epidermal growth factor receptor (EGFR); GLEEVEC® (imatinib mesylate; a protein kinase inhibitor); and ERGAMISOL® (levamisole hydrochloride, Janssen Pharmaceutica Products, LP (Tatesville, NJ); an immunomodulator approved by the FDA in 1990 for use as adjuvant therapy in patients with Dukes stage C colorectal cancer after surgical resection, in combination with 5-fluorouracil).

[0363] For the treatment of lung cancer, exemplary biologics include TARCEVA® (erlotinib HCL, OSI Pharmaceuticals Inc. (Melville, NY; a small molecule designed to target the human epidermal growth factor receptor 1 (HER1) pathway).

[0364] For the treatment of multiple myeloma, exemplary biologics include VELCADE® Velcade (bortezomib, Millennium Pharmaceuticals (Cambridge, Massachusetts); a proteasome inhibitor). Other biologics include THALIDOMID® (thalidomide, Clegene Corporation (Warren County, New Jersey); an immunomodulatory agent that appears to have multiple effects, including the ability to inhibit myeloma cell growth and survival, and anti-angiogenic activity).

[0365] Other exemplary cancer therapeutic antibodies include, but are not limited to, 3F8, abagovomab, adecatumumab, ado-trastuzumab emtansine (KADCYLA®), afutuzumab, alacizumab pegol, alemtuzumab (CAMPATH®, MABCAMPATH®), attumomab pentetate (HYBRI-CEAKER®), anatumomab mafenatox, anrukinzumab (IMA-638), apolizumab, arcitumomab (CEA-SCAN®), atezolizumab (TECENTRIQ®), and avelumab. (BAVENCIO®), bavituximab, bectumomab (LYMPHOSCAN®), belimumab (BENLYSTA®)LYMPHOSTAT-B®, besilesomab (SCINTIMUN®), bevacizumab (AVASTIN®), bivatuzumab mertansine, blinatumomab, brentuximab vedotin, cantuzumab mertansine, capromab pendetide (PROSTASCINT®), catumaxomab (REMOVAB®), CC49, cemiplimab-rwlc (LIBTAYO®), cetuximab (C225, ERBITUX®), citatuzumab bogatox, cixutuzumab, clivatuzumab tetraxetan, conatumumab, dacetuzumab, denosumab (PROLIA®), detumomab, ecromeximab, edrecolomab (PANOREX®), elotuzumab, epitumomab cituxetan, epratuzumab, ertumaxomab (REXOMUN®), etaracizumab, farletuzumab, figitumumab, fresolimumab, galiximab, gemtuzumab ozogamicin (MYLOTARG®), girentuximab, glembatumumab vedotin, ibritumomab tiuxetan,ZEVALIN®, igovomab (INDIMACIS-125®), intetumumab, inotuzumab / ozomicin, ipilimumab, iratumumab, labetuzumab (CEA-CIDE®), lexatumumab, lintuzumab, lucatumumab, lumiliximab, mapatumumab, matuzumab, milatuzumab, minretumomab, mitumomab, nacolomab Tafenatox, naptumomabestafenatox, necitumumab, nimotuzumab (THERACIM®, THERALC®), nofetumomab merpentan (VERLUMA®), ofatumumab (ARZERRA®), olaratumab, oportuzumab monatox, oregovomab (OVAREX®), panitumumab (VECTIBIX®), pemtumomab (THERAGYN®), pertuzumab (OMNITARG®), pintumomab, pritumumab, ramucirumab, ranibumab (LUCENTIS®), rituximab, rituximab (MABTHERA®)RITUXAN®, robatumumab, satumomab pendetide, sibrotuzumab, siltuximab, sontuzumab, tacatuzumab tetraxetan (AFP-CIDE®), tapipramumab paptox, tenatumomab, TGN1412, ticilimumab (tremelimumab), tigatuzumab, TNX-650, tositumomab (BEXXAR®), trastuzumab (HERCEPTIN®), trastuzumab and hyaluronidase-oysk (HERCEPTIN®). HYLECTA®, tremelimumab, tucotuzumab celmoleukin, veltuzumab, volociximab, votumumab (HUMASPECT®), zalutumumab (HUMAX-EGFR®), and zanolimumab (HUMAX-CD4®).

[0366] In other embodiments, the silk fibroin-based microneedles of this disclosure may contain and / or be administered in combination with viral cancer therapeutic agents.Exemplary viral cancer therapeutic agents include, but are not limited to, vaccinia virus (vvDD-CDSR), measles virus expressing carcinoembryonic antigen (CEA), recombinant vaccinia virus (TK-deleted plus GM-CSF), Seneca Valley virus-001, Newcastle disease virus, recombinant modified vaccinia Ankara vaccine expressing Coxsackievirus A21, GL-ONC1, EBNA1 C-terminal / LMP2 chimeric protein, measles virus expressing CEA, G207 oncolytic virus, modified vaccinia virus Ankara vaccine expressing p53, herpes simplex virus type 1 modified with OncoVEX GM-CSF, fowlpox virus vaccine vector, recombinant vaccinia prostate-specific antigen vaccine, human papillomavirus 16 / 18 L1 virus-like particles / AS04 vaccine, and MVA-EBNA1 / LMP2. Inj. Vaccines, Quadrivalent HPV Vaccine, Quadrivalent Human Papillomavirus (Types 6, 11, 16, 18) Recombinant Vaccine (GARDASIL®), Recombinant Fowlpox-CEA(6D) / TRICOM Vaccine; Recombinant Vaccine-CEA(6D)-TRICOM Vaccine, Recombinant Modified Vaccine Ankara-5T4 Vaccine, Recombinant Fowlpox-TRICOM Vaccine, Oncolytic Herpesvirus NV1020, HPV L1 VLP V504 Vaccine, Bivalent Human Papillomavirus (Types 16 and 18) Vaccine (CERVARIX®), Herpes Simplex Virus HF10, Ad5CMV-p53 Gene, Recombinant Vaccine DF3 / MUC1 Vaccine, Recombinant Vaccine-MUC-1 Vaccine, Recombinant Vaccine-TRICOM Vaccine, ALVAC MART-1 vaccine, replication-deficient herpes simplex virus type 1 (HSV-1) vector expressing human pro-endorphin (NP2), wild-type reovirus, reovirus type 3 Dearing (REOLYSIN®), oncolytic virus HSV1716, recombinant modified vaccinia ani (MVA)-based vaccine encoding Epstein-Barr virus target antigen, recombinant fowlpox-prostate-specific antigen vaccine, recombinant vaccinia ani prostate-specific antigen vaccine, recombinant vaccinia ani B7.1 vaccine, rAd-p53 gene, Ad5-Δ24RGD, HPV vaccine 580299, JX-594 (thymidine kinase-deficient vaccinia virus plus GM-CSF), HPV-16 / 18 L1 / AS04, fowlpox virus vaccine vector, vaccinia ani-tyrosinase vaccine, MEDI-517 HPV-16 / 18 VLP AS04 vaccine, adenovirus vector TK99UN containing thymidine kinase of herpes simplex virus, HspE7, FP253 / fludarabine, ALVAC(2) melanoma multiantigen therapy vaccine, ALVAC-hB7.1, canarypox-hIL-12 melanoma vaccine, Ad-REIC / Dkk-3, rAd-IFN SCH 721015, TIL-Ad-INFg, Ad-ISF35 and Coxsackievirus A21 (CVA21, CAVATAK®).In other embodiments, the silk fibroin-based microneedles of this disclosure may comprise latamoxetine (IMLYGIC®) (an FDA-approved treatment for melanoma) and / or may be administered in combination with latamoxetine (IMLYGIC®).

[0367] In other embodiments, the silk fibroin-based microneedles of this disclosure may comprise and / or be administered in combination with a neoantigen vaccine. In some embodiments, the neoantigen vaccine may be prepared as described, for example, in Schumacher et al., Science. 348(6230): 69-74, 2015, which is incorporated herein by reference in its entirety. In some embodiments, the microneedles disclosed herein may be used in methods for identifying neoantigens and / or in methods for preparing neoantigen vaccines. Without wishing to be bound by theory, cancer neoantigens derived from random somatic mutations in tumor tissue are an attractive target type for cancer immunotherapy, including cancer vaccines. Vaccination against tumor-specific neoantigens minimizes the potential induction of central and peripheral tolerance and the risk of autoimmunity. (See, for example, Guo et al., Frontiers in Immunology. Vol. 9 Article 1499, 2018, which is incorporated herein by reference in its entirety). In some embodiments, applying the microneedles of this disclosure to a tumor can expose the subject's immune system to tumor-specific neoantigens and lead to vaccination and immunization against cancers with the same or similar tumor-specific neoantigens.

[0368] In other embodiments, the silk fibroin-based microneedles of this disclosure may comprise and / or be administered in combination with nanomedicines. Exemplary cancer nanomedicines include, but are not limited to, ABRAXANE® (paclitaxel-bound albumin nanoparticles), CRLX101 (camptothecin (CPT) conjugated with a linear cyclodextrin-based polymer), CRLX288 (docetaxel conjugated with a biodegradable polymer poly(lactic-co-glycolic acid)), cytarabine liposomes (liposome Ara-C, DEPOCYT™), daunoxome®, doxorubicin®, citrate-encapsulated doxorubicin®, and PEG anti-VEGF aptamers (MACUGEN®).

[0369] In some embodiments, the silk fibroin-based microneedles of this disclosure may comprise paclitaxel or paclitaxel formulations (e.g., TAXOL®), protein-bound paclitaxel (e.g., ABRAXANE®) and / or may be administered in combination with paclitaxel or paclitaxel formulations (e.g., TAXOL®) or protein-bound paclitaxel (e.g., ABRAXANE®). Exemplary paclitaxel formulations include, but are not limited to, albumin-bound paclitaxel nanoparticles (ABRAXANE®, sold by Abraxis Bioscience), docosahexaenoic acid-bound paclitaxel (DHA-paclitaxel, docosahexaenoic acid paclitaxel, sold by Protarga), polyglutamate-bound paclitaxel (PG-paclitaxel, polyglutamate paclitaxel, CT-2103, sold by XYOTAX, sold by Cell Therapeutic), tumor-activating prodrugs (TAP), ANG105 (Angiopep-2 bound to three molecules of paclitaxel, sold by ImmunoGen), and paclitaxel-EC-1 (paclitaxel bound to erbB2-recognition peptide EC-1; see Li et al.). Biopolymers (2007) 87:225-230) and glucose-conjugated paclitaxel (e.g., 2'-paclitaxel methyl 2-pyranose glucosuccinate (see, for example, Liu et al.) , Bioorganic&Medicinal ChemistryLetters (2007) 17:617-620).

[0370] Exemplary RNAi and antisense RNA agents for treating cancer include, but are not limited to, CALAA-01, siG12DLODER (Local Drug EluteR), and ALN-VSP02.

[0371] Other cancer treatment agents include, but are not limited to, cytokines (e.g., adefovir (IL-2, interleukin-2, PROLEUKIN®), alpha interferon (IFN-α, interferon α, INTRON® A (interferon α-2b), ROFERON-A® (interferon α-2a)), epoetin alfa (PROCRIT®), filgrastim (G-CSF, granulocyte colony-stimulating factor, NEUPOGEN®), GM-CSF (granulocyte-macrophage colony-stimulating factor, sammostatin, LEUKINE™), IL-11 (interleukin-11, olprene interleukin, NEUMEGA®), and interferon α-2b (PEG conjugate). (PEG interferon, PEG-INTRON™, and PEG-Filagraxetine (NEULASTA™)), hormone therapy agents (e.g., aminoglutethimide (CYTADREN®), anastrozole (ARIMIDEX®), bicalutamide (CASODEX®), exemestane (AROMASIN®), fluorometholone (HALOTESTIN®), flutamide (EULEXIN®), fulvestrant (FASLODEX®), goserelin (ZOLADEX®), letrozole (FEMARA®), leuprolide (ELIGARD™, LUPRON®, LUPRON DEPOT®, VIADUR™), medroxyprogesterone acetate (MEGACE®), niglutethimide (ANANDRON®, NILANDRON®), octreotide (octreotide acetate, SANDOSTATIN®, SANDOSTATIN) LAR®, raloxifene (EVISTA®), romistastatin (NPLATE®), tamoxifen (NOVALDEX®), and toremifene (FARESTON®), phospholipase A2 inhibitors (e.g., anagrelide (AGRYLIN®), biological response modifiers (e.g., BCG (THERACYS®, TICE®), and dapoxetine alpha (ARANESP®), targeted therapy agents (e.g., bortezomib (VELCADE®), dasatinib (SPRYCEL™), dentine interleukin-toxin conjugate (ONTAK®), erlotinib (TARCEVA®), everolimus (AFINITOR®), gefitinib (IRESSA®), imatinib mesylate (STI-571, GLEEVEC™), lapatinib (TYKERB®), sorafenib (NEXAVAR®), and SU11248 (sunitinib, SUTENT®), immunomodulatory and anti-angiogenic agents (e.g., CC-5013 (lenalidomide, REVLIMID® and thalidomide (THALOMID®)), glucocorticoids (e.g.,Cortisone (hydrocortisone, hydrocortisone sodium phosphate, hydrocortisone sodium succinate, ALA-CORT®, HYDROCORTACETATE®, hydrocortisone phosphate LANACORT®, SOLU-CORTEF®), decatelon (dexamethasone, dexamethasone acetate, dexamethasone sodium phosphate, DEXASONE®, DIODEX®, HEXADROL®, MAXIDEX®), methylprednisolone (6-methylprednisolone, methylprednisolone acetate, methylprednisolone sodium succinate, DURALONE®, MEDRALONE®, MEDROL®, M-PREDNISOL®, SOLU-MEDROL®), prednisolone (DELTA-CORTEF®, ORAPRED®, PEDIAPRED®, PRELONE®), and prednisone (DELTASONE®, LIQUID®). PRED®, METICORTEN®, ORASONE®) and bisphosphonates (e.g., pamidronate (AREDIA®) and zoledronic acid (ZOMETA®)).

[0372] In some embodiments, the silk fibroin-based microneedles of this disclosure may comprise a tyrosine kinase inhibitor (e.g., a receptor tyrosine kinase (RTK) inhibitor) and / or may be administered in combination with a tyrosine kinase inhibitor (e.g., a receptor tyrosine kinase (RTK) inhibitor). Exemplary tyrosine kinase inhibitors include, but are not limited to, epidermal growth factor (EGF) pathway inhibitors (e.g., epidermal growth factor receptor (EGFR) inhibitors), vascular endothelial growth factor (VEGF) pathway inhibitors (e.g., anti-VEGF antibodies, VEGF scavengers, vascular endothelial growth factor receptor (VEGFR) inhibitors (e.g., VEGFR-1 inhibitors, VEGFR-2 inhibitors, VEGFR-3 inhibitors)), platelet-derived growth factor (PDGF) pathway inhibitors (e.g., platelet-derived growth factor receptor (PDGFR) inhibitors (e.g., PDGFR-β inhibitors)), RAF-1 inhibitors, KIT inhibitors, and RET inhibitors. In some implementation schemes, the anticancer agents used in combination with AHCM agents are selected from: axitinib (AG013736), bosutinib (SKI-606), cidinib (RECENTINTM, AZD2171), dasatinib (SPRYCEL®, BMS-354825), erlotinib (TARCEVA®), gefitinib (IRESSA®), imatinib (Gleevec®, CGP57148B, STI-571), lapatinib (TYKERB®, TYVERB®), lenatatinib (CEP-701), neratinib (HKI-272), nilotinib (TASIGNA®), semaxanib (semaxinib, SU5416), sunitinib (SUTENT®,SU11248), Tocinivumab (PALLADIA®), Vandetanib (ZACTIMA®, ZD6474), Vatalani (PTK787, PTK / ZK), Trastuzumab (HERCEPTIN®), Bevacizumab (AVASTIN®), Rituximab (RITUXAN®), Cetuximab (ERBITUX®), Panitumumab (VECTIBIX®), Ranibizumab (Lucentis®), Nilotinib (TASIGNA®), Sorafenib (NEXAVAR®), Alemazumab (CAMPATH®), Gemtuzumab Ozomicin (MYLOTARG®), ENMD-2076, PCI-32765, AC220, Dovitinib Lactate (TKI258, CHIR-258), BIBW 2992 (TOVOKTM), SGX523, PF-04217903, PF-02341066, PF-299804, BMS-777607, ABT-869, MP470, BIBF 1120 (VARGATEF®), AP24534, JNJ-26483327, MGCD265, DCC-2036, BMS-690154, CEP-11981, Tevozani (AV-951), OSI-930, MM-121, XL-184, XL-647, XL228, AEE788, AG-490, AST-6, BMS-599626, CUDC-101, PD153035, Peritoltinib (EKB-569), Vandetanib (zactima), WZ3146, WZ4002, WZ8040, ABT-869 (Linevanivar), AEE788, AP24534 (Punatinib), AV-951 (Tevozani), Axitinib, BAY 73-4506 (Regorafenib), Brinib alanine (BMS-582664), Brinib (BMS-540215), Sildenafil (AZD2171), CHIR-258 (Dovitinib), CP 673451, CYC116, E7080, Ki8751, Macitinib (AB1010), MGCD-265, Motisanib diphosphate (AMG-706), MP-470, OSI-930, Pazopanib hydrochloride, PD173074, Sorafenib tosylate (Bay 43-9006), SU 5402, TSU-68 (SU6668), Vatalanib, XL880 (GSK1363089, EXEL-2880). The selected tyrosine kinase inhibitors were sunitinib, erlotinib, gefitinib, or sorafenib. In one implementation, the tyrosine kinase inhibitor is sunitinib.

[0373] In some embodiments, the silk fibroin-based microneedles of this disclosure may comprise one or more of the following and / or be administered in combination with one or more of the following: an anti-angiogenic agent, or a vascular targeting agent or a vascular disruptor. Exemplary anti-angiogenic agents include, but are not limited to, vascular endothelial growth factor (VEGF) inhibitors (e.g., anti-VEGF antibodies (e.g., bevacizumab); VEGF receptor inhibitors (e.g., itraconazole); inhibitors of cell proliferation and / or endothelial cell migration (e.g., carboxytriazole, TNP-470); inhibitors of angiogenesis stimulants (e.g., suramin), etc. Vascular targeting agents (VTAs) or vascular disruptors (VDAs) are designed to damage the vascular system (blood vessels) of cancerous tumors that lead to central necrosis (as in, for example, Thorpe, PE (2004)). Clin. Cancer Res (Reviewed in Vol. 10:415-427). VTAs can be small molecules. Exemplary small molecule VTAs include, but are not limited to, microtubule-destabilized drugs (e.g., cobustatin A-4 phosphate disodium (CA4P), ZD6126, AVE8062, Oxi 4503); and vadimezan (ASA404).

[0374] In some implementations, the anticancer agents described herein may be formulated in sustained-release particles.

[0375] Immunomodulators Checkpoint inhibitors In some embodiments, the silk fibroin-based microneedles of this disclosure may comprise and / or be administered in combination with immune checkpoint inhibitors. For example, in some embodiments, the silk fibroin-based microneedles may be used to locally administer therapeutic agents (e.g., anticancer agents and / or immunomodulators) to a subject's tumor in combination with systemic administration (e.g., by injection) of a checkpoint inhibitor (e.g., an antiPD1 antibody).

[0376] In the implementation, immune checkpoint inhibitors inhibit checkpoint molecules. Exemplary checkpoint molecules include, but are not limited to, CTLA4, PD1, PD-L1, PD-L2, TIM3, LAG3, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), BTLA, KIR, MHC class I, MHC class II, GAL9, VISTA, BTLA, TIGIT, LAIR1, and A2aR. See, for example, Pardoll. Nat. Rev. Cancer 12.4(2012):252-64, which is incorporated herein by reference.

[0377] In some implementations, the immune checkpoint inhibitor is a PD-1 inhibitor, such as an anti-PD-1 antibody like nivolumab, pembrolizumab, or pildizumab. Nivolumab (also known as MDX-1106, MDX-1106-04, ONO-4538, or BMS-936558) is a fully human IgG4 monoclonal antibody that specifically inhibits PD-1. See, for example, US 8,008,449 and WO2006 / 121168. Pembrolizumab (also known as rambombab, MK-3475, MK03475, SCH-900475, or KEYTRUDA®; Merck) is a humanized IgG4 monoclonal antibody that binds to PD-1. (See, for example, Hamid, O. et al., (2013)) New England Journal of Medicine 369 (2): 134–44; US 8,354,509 and WO2009 / 114335). Pildizumab (also known as CT-011 or Cure Tech) is a humanized IgG1k monoclonal antibody that binds to PD1. (See, for example, WO2009 / 101611). In one embodiment, the PD-1 inhibitor is an antibody molecule having a sequence substantially identical or similar to that of nivolumab, pembrolizumab, or pitizumab, for example, a sequence at least 85%, 90%, 95% identical or higher to that of nivolumab, pembrolizumab, or pitizumab. Additional anti-PD1 antibodies, such as AMP514 (Amplimmune), are described, for example, in US 8,609,089, US 2010028330 and / or US 20120114649.

[0378] In some embodiments, the PD-1 inhibitor is an immunoadhesin, for example, an immunoadhesin comprising an extracellular / PD-1 binding portion of a PD-1 ligand (e.g., PD-L1 or PD-L2) fused to a constant region (e.g., the Fc region of an immunoglobulin). In embodiments, the PD-1 inhibitor is AMP-224 (B7-DCIg, as described, for example, in WO2011 / 066342 and WO2010 / 027827), which is a PD-L2 Fc fusion soluble receptor that blocks the interaction between B7-H1 and PD-1.

[0379] In some embodiments, the immune checkpoint inhibitor is a PD-L1 inhibitor, such as an antibody molecule. In some embodiments, the PD-L1 inhibitor is YW243.55.S70, MPDL3280A, MEDI-4736, MSB-0010718C, or MDX-1105. In some embodiments, the anti-PD-L1 antibody is MSB0010718C (also known as A09-246-2; MerckSerono), which is a monoclonal antibody that binds to PD-L1. Exemplary humanized anti-PD-L1 antibodies are described, for example, in WO2013 / 079174. In one embodiment, the PD-L1 inhibitor is an anti-PD-L1 antibody, such as YW243.55.S70. The YW243.55.S70 antibody is described, for example, in WO 2010 / 077634. In one embodiment, the PD-L1 inhibitor is MDX-1105 (also known as BMS-936559), as described, for example, in WO2007 / 005874. In one embodiment, the PD-L1 inhibitor is MDPL3280A (Genentech / Roche), a human Fc-optimized IgG1 monoclonal antibody against PD-L1. See, for example, U.S. Patent No. 7,943,743 and U.S. Publication No. 20120039906. In one embodiment, the PD-L1 inhibitor is an antibody molecule having a sequence substantially identical or similar to that of YW243.55.S70, MPDL3280A, MEDI-4736, MSB-0010718C, or MDX-1105, for example, having a sequence that is at least 85%, 90%, or 95% identical or higher to that of YW243.55.S70, MPDL3280A, MEDI-4736, MSB-0010718C, or MDX-1105.

[0380] In the implementation scheme, the immune checkpoint inhibitor is a PD-L2 inhibitor, such as AMP-224 (which is a PD-L2 Fc fusion soluble receptor that blocks the interaction between PD1 and B7-H1). See, for example, WO2010 / 027827 and WO2011 / 066342.

[0381] In one embodiment, the immune checkpoint inhibitor is a LAG-3 inhibitor, such as an anti-LAG-3 antibody molecule. In another embodiment, the anti-LAG-3 antibody is BMS-986016 (also known as BMS986016; Bristol-Myers Squibb). BMS-986016 and other humanized anti-LAG-3 antibodies are described, for example, in US 2011 / 0150892, WO2010 / 019570, and WO2014 / 008218.

[0382] In the implementation scheme, the immune checkpoint inhibitor is a TIM-3 inhibitor, such as an anti-TIM3 antibody molecule, as described in, for example, U.S. Patent Nos. 8,552,156, WO 2011 / 155607, EP 2581113 and U.S. Publication No. 2014 / 044728.

[0383] In the implementation, the immune checkpoint inhibitor is a CTLA-4 inhibitor, such as an anti-CTLA-4 antibody molecule. Exemplary anti-CTLA4 antibodies include trimemumab (an IgG2 monoclonal antibody from Pfizer, formerly known as tesimumab, CP-675,206); and ipilimumab (also known as MDX-010, CAS No. 477202-00-9). Other exemplary anti-CTLA-4 antibodies are described, for example, in U.S. Patent No. 5,811,097.

[0384] TLR agonists In some embodiments, the silk fibroin-based microneedles of this disclosure may contain a Toll-like receptor (TLR) agonist and / or may be administered in combination with a Toll-like receptor (TLR) agonist.

[0385] TLRs are a family of pattern recognition receptors initially identified as sensors of the innate immune system that recognize microbial pathogens. In humans, TLRs include TLR-1, TLR-2, TLR-3, TLR-4, TLR-5, TLR-6, ​​TLR-7, TLR-8, TLR-9, and TLR-10. TLR-1, TLR-2, TLR-4, TLR-5, and TLR-6 are expressed on the cell surface, while TLR-3, TLR-7 / 8, and TLR-9 are expressed in the ER compartment. Human dendritic cell subsets can be identified based on different TLR expression patterns. The bone marrow or “normal” subset of human dendritic cells expresses TLRs 1–8, while the plasmacytoid subset of dendritic cells expresses only TLR-7 and TLR-9. Ligands binding to TLRs trigger cascades of intracellular signaling pathways, inducing the production of factors involved in inflammation and immunity. Upon stimulation, bone marrow subsets and plasma cell-like subsets of human dendritic cells lead to the initiation and activation of antigen-specific CD4+ and CD8+ T cells of NK cells and T cells, respectively.

[0386] In some embodiments, the TLR agonist is selected from one or more of the following: TLR-1 agonist, TLR-2 agonist, TLR-3 agonist, TLR-4 agonist, TLR-5 agonist, TLR-6 agonist, TLR-7 agonist, TLR-8 agonist, TLR-9 agonist, TLR-10 agonist, TLR-1 / 2 agonist, TLR-2 / 6 agonist, or TLR-7 / 8 agonist. In one embodiment, the TLR agonist is a TLR7 agonist.

[0387] In some embodiments, the TLR agonist is imiquimod or 3-(2-methylpropyl)-3,5,8-triazatricyclo[7.4.0.02,6]tridecyl-1(9),2(6),4,7,10,12-hexen-7-amine. Imiquimod or 3-(2-methylpropyl)-3,5,8-triazatricyclo[7.4.0.02,6]tridecyl-1(9),2(6),4,7,10,12-hexen-7-amine can bind to and activate TLR-7 and / or TLR-8.

[0388] In some implementations, the TLR agonist is 852A. 852A is used, for example, by Inglefield et al. J Interferon Cytokine Res. It is disclosed in 2008; 28(4):253-63. The 852A can bind to and activate TLR-7 and / or TLR-8.

[0389] In some implementations, the TLR agonist is BCG (Bacillus Calmette-Guérin). BCG can bind to and activate TLR-9.

[0390] In some implementations, the TLR agonist is EMD 120108. EMD 120108 is a synthetic oligonucleotide containing a phosphate thioester oligodeoxynucleotide. EMD 1201081 can bind to and activate TLR-9, for example, in monocytes / macrophages, plasmacytoid dendritic cells (DCs), and B cells, thereby initiating immune signaling pathways, activating B cells, and inducing the production of helper T cell cytokines.

[0391] In some implementations, the TLR agonist is IMO-2055. IMO-2055 is a synthetic oligonucleotide containing an unmethylated CpG dinucleotide. By mimicking the unmethylated CpG sequence in bacterial DNA, IMO-2055 can bind to and activate TLR-9, for example, in monocytes / macrophages, plasmacytoid dendritic cells (DCs), and B cells, thereby initiating immune signaling pathways, activating B cells and DCs, and inducing the production of helper T cell cytokines.

[0392] Other exemplary TLR agonists that can be used in combination include, for example, TLR-1 / 2 agonists (e.g., Pam3Cys), TLR-2 agonists (e.g., CFA, MALP2, Pam2Cys, FSL-1, or Hib-OMPC), TLR-3 agonists (e.g., polyinosinic-polycytidylic acid (Poly I:C), polyadenosine-polyuridine (poly AU), polyinosinic-polycytidylic acid stabilized with poly-L-lysine and carboxymethyl cellulose (Hiltonol®), TLR-4 agonists (e.g., monophospholipid A (MPL), LPS, sialyl-Tn (STn)), TLR-5 agonists (e.g., bacterial flagellin), TLR-7 agonists (e.g., imiquimod), TLR-7 / 8 agonists (e.g., requimod or loxolibin), and TLR-9 agonists (e.g., unmethylated CpG dinucleotides (CpG-ODN)).

[0393] In another embodiment, the TLR agonist is used in combination with the GITR agonist, as described, for example, in WO2004 / 060319 and International Publication No. WO2014 / 012479.

[0394] STING agonist In some embodiments, the silk fibroin-based microneedles of this disclosure may contain a STING agonist and / or may be administered in combination with a STING agonist.

[0395] In some embodiments, the STING agonist is a cyclic dinucleotide, such as a cyclic dinucleotide containing a purine or pyrimidine nucleobase (e.g., adenosine, guanine, uracil, thymine, or cytosine nucleobase). In some embodiments, the cyclic dinucleotide contains the same or different nucleobases.

[0396] In some embodiments, the STING agonist comprises an adenosine or guanosine nucleobase. In some embodiments, the STING agonist comprises one adenosine nucleobase and one guanosine nucleobase. In some embodiments, the STING agonist comprises two adenosine nucleobases or two guanosine nucleobases.

[0397] In some embodiments, the STING agonist comprises a modified cyclic dinucleotide, for example, comprising a modified nucleobase, a modified ribose, or a modified phosphate bond. In some embodiments, the modified cyclic dinucleotide comprises a modified phosphate bond, for example, a thiophosphate ester.

[0398] In some embodiments, the STING agonist comprises a cyclic dinucleotide (e.g., a modified cyclic dinucleotide) having a 2', 5', or 3', 5' phosphate bond. In some embodiments, the STING agonist comprises a cyclic dinucleotide (e.g., a modified cyclic dinucleotide) having Rp or Sp stereochemistry around the phosphate bond.

[0399] In some embodiments, the STING agonist is Rp,Rp-dithio2',3'c-di-AMP (e.g., Rp,Rp-dithioc-[A(2',5')pA(3',5')p]) or a cyclic dinucleotide analog thereof. In some embodiments, the STING agonist is a compound described in U.S. Patent Publication No. US2015 / 0056224 (e.g., Figure 2 Compounds in c, for example, compound 21 or compound 22). In some embodiments, the STING agonist is c-[G(2',5')pG(3',5')p], its dithioribose O-substituted derivative, or a compound specified in PCT Publications WO 2014 / 189805 and WO 2014 / 189806. Figure 4 The compounds described in [the document]. In some embodiments, the STING agonist is c-[A(2',5')pA(3',5')p] or an O-substituted derivative of its dithioribose, or is a compound as described in PCT Publications WO 2014 / 189805 and WO 2014 / 189806. Figure 5 The compounds described in [the document]. In some embodiments, the STING agonist is c-[G(2',5')pA(3',5')p] or an O-substituted derivative of its dithioribose, or is a compound as described in PCT Publications WO 2014 / 189805 and WO 2014 / 189806. Figure 5 The compounds depicted in Figure 7 of PCT Publication No. WO 2014 / 189806. In some embodiments, the STING agonist is 2'-O-propynyl-cyclic-[A(2',5')pA(3',5')p] (2'-O-propynyl-ML-CDA) or the compound depicted in Figure 7 of PCT Publication No. WO 2014 / 189806.

[0400] Other exemplary STING agonists are disclosed, for example, in PCT Publications WO 2014 / 189805 and WO 2014 / 189806 and U.S. Publication 2015 / 0056225.

[0401] RIG agonist In some embodiments, the silk fibroin-based microneedles of this disclosure may comprise a RIG agonist (i.e., an agonist of retinoic acid-induced gene I (RIG-I) encoded by the gene DDX58) and / or may be administered in combination with said RIG agonist. Exemplary RIG agonists are described in Elion et al., Oncotarget. 9(48): 29007–29017, 2018, which is incorporated herein by reference in its entirety.

[0402] Cytokines In some embodiments, the silk fibroin-based microneedles of this disclosure may contain cytokines and / or be administered in combination with cytokines.

[0403] Cytokines are typically polypeptides that influence cellular activity, for example, through signal transduction pathways. Therefore, cytokines are useful and can be associated with receptor-mediated signaling, which transmits signals from outside the cell membrane to regulate intracellular responses. Cytokines are protein signaling compounds and mediators of the immune response. They control many different cellular functions, including proliferation, differentiation, and cell survival / apoptosis; cytokines also participate in several pathophysiological processes, including viral infections and autoimmune diseases. Cytokines are synthesized by various cells of the innate immune system (monocytes, macrophages, dendritic cells) and the adaptive immune system (T cells and B cells) in response to various stimuli. Cytokines can be divided into two groups: pro-inflammatory and anti-inflammatory. Pro-inflammatory cytokines, including IFNγ, IL-1, IL-6, and TNF-α, are mainly derived from innate immune cells and Th1 cells. Anti-inflammatory cytokines, including IL-10, IL-4, IL-13, and IL-5, are synthesized by Th2 immune cells.

[0404] Therefore, in some embodiments, the cytokine molecule is an interleukin or a variant thereof, such as a functional variant. In some embodiments, the interleukin is a pro-inflammatory interleukin. In some embodiments, the interleukin is selected from interleukin-2 (IL-2), interleukin-12 (IL-12), interleukin-15 (IL-15), interleukin-18 (IL-18), interleukin-21 (IL-21), interleukin-7 (IL-7), or interferon-γ. In some embodiments, the cytokine molecule is a pro-inflammatory cytokine. In one embodiment, the cytokine molecule is IL-18.

[0405] Cytokines can be wild-type (e.g., wild-type recombinants) or genetically engineered (e.g., to introduce one or more mutations). In some embodiments, the cytokine is an engineered cytokine, such as an engineered interleukin. In some embodiments, the engineered cytokine contains one or more mutations, for example, to provide biological properties different from wild-type variants. For example, the cytokine molecule can be an engineered “anti-decoy” interleukin (e.g., anti-decoy IL-18), such as those described by Zhou et al. Nature (2020) 583:609-614, which is incorporated herein by reference in its entirety. In some embodiments, the cytokine is an engineered interleukin (e.g., engineered IL-2 or engineered IL-18).

[0406] In some embodiments, cytokines are engineered to improve one or more pharmacokinetic properties. In some embodiments, the engineered cytokines comprise cytokines fused to another molecule (e.g., an antibody), for example to increase the serum half-life of the cytokines. In some embodiments, cytokines are fused to long-half-life proteins or protein domains (e.g., Fc fusion, transferrin [Tf] fusion, or albumin fusion). In some embodiments, cytokines are fused to inert peptides (e.g., XTEN or recombinant PEG (rPEG); homoamino acid polymers (HAP; e.g., via HAP fusion); proline-alanine-serine polymers (PAS; e.g., via PAS fusion); or elastin-like peptides (ELP; e.g., via ELP fusion). In some embodiments, cytokines are conjugated to repeating chemical moieties (e.g., polymers, such as PEG-fused PEG; or hyaluronic acid), for example to increase the hydrodynamic radius of the cytokines. In some embodiments, the negative charge of the cytokine is increased, for example, by polysialylation of the cytokine, or by fusing a negatively charged, highly sialylated peptide (e.g., a carboxyl-terminal peptide [CTP; β-chain of human chorionic gonadotropin (CG)]) to the cytokine. In some embodiments, the cytokine is non-covalently bound to a long-half-life protein such as HSA, human IgG, or transferrin. In some embodiments, the cytokine is chemically conjugated to a long-half-life protein such as human IgG, the Fc moiety, or HSA. Methods for preparing and using fusion proteins and similarly modified proteins (e.g., to extend half-life) have been described (see, for example, Strohl et al., BioDrugs (2015) 29:215-239; and the references cited therein).

[0407] Engineered cytokines (e.g., engineered interleukins) can be produced by known methods, such as through directed evolution techniques (see, for example, Zhou et al., above). Examples of engineered cytokines (e.g., engineered interleukins) have been described (see, for example, WO 2012 / 107417; WO 2009 / 061853; U.S. Patent No.: 9,580,486; Minsahwi et al., Front Immunol. 2020 (11);1794; Tang et al., Cytokine X (2019) 100001; Mitra et al., Immunity (2015) 42:826-838; Casadesús et al., OncoImmunology (2020) 9: 1770565; Zhou et al., see above; each is incorporated herein by reference in its entirety.

[0408] To avoid being bound by theory, engineered cytokines (e.g., "anti-decoy" interleukins, such as anti-decoy IL-18) may possess advantageous properties compared to their wild-type counterparts. For example, anti-decoy IL-18 may retain signal transduction potential but remain unaffected by inhibition from IL-18-binding protein (IL-18BP). In some embodiments, engineered cytokines exhibit improved stability (e.g., improved temperature-dependent stability, pH-dependent stability, or both) compared to wild-type cytokines. In some embodiments, engineered cytokines have improved serum half-life relative to wild-type cytokines. In some embodiments, engineered cytokines exhibit altered affinity for receptors (e.g., enhanced affinity) unlike wild-type cytokines. In some embodiments, engineered cytokines exhibit lower toxicity relative to wild-type cytokines.

[0409] In some embodiments, the cytokine is a single-chain cytokine. In some embodiments, the cytokine is a multi-chain cytokine (e.g., the cytokine comprises two or more polypeptide chains). An exemplary multi-chain cytokine is IL-12.

[0410] Examples of useful cytokines include, but are not limited to, GM-CSF, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-18, IL-21, IFN-α, IFN-β, IFN-γ, MIP-1α, MIP-1β, TGF-β, TNF-α, and TNFβ. In one embodiment, the cytokine of the multispecific or multifunctional peptide is a cytokine selected from GM-CSF, IL-2, IL-7, IL-8, IL-10, IL-12, IL-15, IL-18, IL-21, IFN-α, IFN-γ, MIP-1α, MIP-1β, and TGF-β. In one embodiment, the cytokine of the multispecific or multifunctional peptide is a cytokine selected from GM-CSF, IL-2, IL-7, IL-8, IL-10, IL-12, IL-15, IL-21, IFN-α, IFN-γ, MIP-1α, MIP-1β, and TGF-β. In another embodiment, the cytokine of the multispecific or multifunctional peptide is a cytokine selected from IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IFN-α, and IFN-γ. In yet another embodiment, the cytokine of the multispecific or multifunctional peptide is a cytokine selected from IL-2, IL-7, IL-10, IL-12, IL-15, IFN-α, and IFN-γ. In some embodiments, the cytokine is mutated to remove N- and / or O-glycosylation sites. Elimination of glycosylation can increase the homogeneity of the product available in recombinant production.

[0411] In one embodiment, the cytokine is IL-2. In a specific embodiment, the IL-2 cytokine can induce one or more cellular responses selected from the following: proliferation in activated T lymphocytes, differentiation in activated T lymphocytes, cytotoxic T cell (CTL) activity, proliferation in activated B cells, differentiation in activated B cells, proliferation in natural killer (NK) cells, differentiation in NK cells, cytokine secretion by activated T cells or NK cells, and antitumor cytotoxicity of NK / lymphocyte-activated killer cells (LAK).

[0412] In another embodiment, the cytokine is IL-12. In a specific embodiment, the IL-12 cytokine is a single-chain IL-12 cytokine. In one embodiment, the IL-12 cytokine can induce one or more cellular responses selected from: proliferation in NK cells, differentiation in NK cells, proliferation in T cells, and differentiation in T cells.

[0413] In another embodiment, the cytokine is IL-10. In yet another specific embodiment, the IL-10 cytokine is a monomeric IL-10 cytokine. In one embodiment, the IL-10 cytokine can induce one or more cellular responses selected from the following: inhibition of cytokine secretion, inhibition of antigen presentation by antigen-presenting cells, reduction of oxygen free radical release, and inhibition of T cell proliferation.

[0414] In one specific embodiment, the IL-15 cytokine is a mutant IL-15 cytokine with reduced binding affinity to the α-subunit of the IL-15 receptor. Unwilling to be bound by theory, the reduced binding affinity of the mutant IL-15 peptide to the α-subunit of the IL-15 receptor to systemic fibroblasts results in a decreased ability to bind, leading to improved pharmacokinetic and toxicological characteristics compared to the wild-type IL-15 peptide. In one embodiment, the IL-15 cytokine can induce one or more cellular responses selected from: proliferation in activated T lymphocytes, differentiation in activated T lymphocytes, cytotoxic T cell (CTL) activity, proliferation in activated B cells, differentiation in activated B cells, proliferation in natural killer (NK) cells, differentiation in NK cells, cytokine secretion from activated T cells or NK cells, and NK / lymphocyte-activated killer cell (LAK) antitumor cytotoxicity.

[0415] In another embodiment, the cytokine is interleukin-18 (IL-18). In one embodiment, the cytokine is an IL-18 variant polypeptide. In one embodiment, the IL-18 cytokine is an anti-decoy IL-18 (see, for example, U.S. Patent Publication No. 2019 / 0070262; and Zhou et al., see above, each of which is incorporated herein by reference in its entirety). In one embodiment, the IL-18 cytokine can induce one or more cellular responses selected from: proliferation in NK cells, differentiation in NK cells, proliferation in T cells, differentiation in T cells, and mimicry of lymphocytes (e.g., innate lymphocytes). Not wishing to be bound by theory, IL-18 can be used as an effective immunotherapeutic agent and is well tolerable in humans (see, for example, Robertson et al., Clin. Cancer Res.(2006) 12:4265-4273). In some embodiments, IL-18 (e.g., anti-decoy IL-18) increases the population of precursor T cells (e.g., CD8 T cells) expressing transcription factors with anti-tumor functions (e.g., Tcf1). In some embodiments, IL-18 (e.g., anti-decoy IL-18) promotes the differentiation of T cells into a highly active, multifunctional effector phenotype. In some embodiments, IL-18 (e.g., anti-decoy IL-18) reduces the incidence of exhausted CD8+ T cells (e.g., those expressing transcriptional regulators of exhausted TOX). In some embodiments, IL-18 (e.g., anti-decoy IL-18) enhances the activity and / or maturation of NK cells.

[0416] In some embodiments, the cytokine is the anti-decoy interleukin IL-18. In some embodiments, the cytokine is an IL-18 variant peptide, wherein the IL-18 variant peptide specifically binds to the IL-18 receptor (IL-18R) and, compared to wild-type (WT) IL-18, the IL-18 variant peptide contains at least one mutation and exhibits significantly reduced binding to the IL-18 binding protein (IL-18BP). In some embodiments, the cytokine is an IL-18 variant polypeptide relative to the wild-type IL-18 sequence, such as SEQ ID NO: 30 of US2019 / 0070262, comprising at least one mutation selected from the following: Y1X, L5X, K8X, M51X, K53X, S55X, Q56X, P57X, G59X, M60X, E77X, Q103X, S105X, D110X, N111X, M113X, V153X, and N155X. In some embodiments, the cytokine is an IL-18 mutant polypeptide relative to the wild-type IL-18 sequence, such as SEQ ID NO: 30 of US2019 / 0070262. 30, which contains at least one mutation selected from the following: Y1H, Y1R, L5H, L5I, L5Y, K8Q, K8R, M51T, M51K, M51D, M51N, M51E, M51R, K53R, K53G, K53S, K53T, S55K, S55R, Q56E, Q56A, Q56R, Q56V, Q56G, Q56K, Q56L, P57L, P57G, P57A, P57K, G59T, G59A, M60K, M60Q, M60R, M60L, E77 D, Q103E, Q103K, Q103P, Q103A, Q103R, S105R, S105D, S105K, S105N, S105A, D110H, D110K, D110N, D110Q, D110E, D110S, D110G, N111H, N111Y, N111D, N111R, N111S, N111G, M113V, M113R, M113T, M113K, V153I, V153T, V153A, N155K, and N155H. In some embodiments, the cytokine is an IL-18 variant polypeptide relative to the wild-type IL-18 sequence, such as SEQ ID NO: 30 of US2019 / 0070262, which contains at least six mutations selected from the following: Y1X, L5X, K8X, M51X, K53X, 555X, Q56X, P57X, G59X, M60X, E77X, Q103X, S105X, D110X, N111X, M113X, V153X, and N155X.In some embodiments, the cytokine is an IL-18 variant polypeptide relative to the wild-type IL-18 sequence, such as SEQ ID NO:30 of US2019 / 0070262, which contains mutations at positions M51, K53, Q56, D110, and N111. In some embodiments, the cytokine is an IL-18 variant polypeptide sequence relative to the wild-type IL-18 sequence, such as SEQ ID NO: 30 of US2019 / 0070262, which contains the following five mutations: (i) M51E, M51R, M51K, M51T, M51D, or M51N; (ii) K53G, K53S, K53T, or K53R; (iii) Q56G, Q56R, Q56L, Q56E, Q56A, Q56V, or Q56K; (iv) D110S, D110N, D110G, D110K, D110H, D110Q, or D110E; and (v) N111G, N111R, N111S, N111D, N111H, or N111Y. In some embodiments, the cytokine is an IL-18 variant polypeptide relative to the wild-type IL-18 sequence, such as SEQ ID NO: 30 of US2019 / 0070262, which also contains mutations at positions P57 and M60. In some embodiments, the cytokine is an IL-18 variant polypeptide relative to the wild-type IL-18 sequence, such as SEQ ID NO: 30 of US2019 / 0070262, which contains the following seven mutations: (i) M51E, M51R, M51K, M51T, M51D, or M51N; (ii) K53G, K53S, K53T, or K53R; (iii) Q56G, Q56R, Q56L, Q56E, Q56A, Q56V, or Q56K; (iv) D110S, D110N, D110G, D110K, D110H, D110Q, or D110E; (v) N111G, N111R, N111S, N111D, N111H, or N111Y; (vi) P57A, P57L, P57G, or P57K; and (vii) M60L, M60R, M60K, or M600. In some embodiments, the cytokine is an IL-18 variant polypeptide sequence comprising the polypeptide sequence described in US2019 / 0070262 (e.g., SEQ ID NO.: 34-59, 60-72, 73-91, 191-193, or fragments thereof).

[0417] Unwilling to be bound by theory, IL-18 variants (e.g., anti-decoy IL-18) can evade IL-18BP and further enhance innate antitumor immunity by stimulating NK cell activity and / or enhancing NK cell maturation. They can also exert antitumor immunity against tumors resistant to conventional immune checkpoint blockade therapies (e.g., anti-PD-1 plus anti-CLTA-4) due to loss of MHC class I surface expression.

[0418] Mutant cytokine molecules that can be used as effector parts can be prepared by deletion, substitution, insertion, or modification using genetic or chemical methods known in the art. Genetic methods may include site-specific mutagenesis encoding DNA sequences, PCR, gene synthesis, etc. Correct nucleotide changes can be verified, for example, by sequencing. Substitution or insertion may involve both natural and non-natural amino acid residues. Amino acid modification includes known chemical modification methods such as adding or removing glycosylation sites or attaching carbohydrates.

[0419] In one implementation, the cytokine is GM-CSF. In a specific implementation, the GM-CSF cytokine can induce the proliferation and / or differentiation of granulocytes, monocytes, or dendritic cells.

[0420] In one embodiment, the cytokine is IFN-α. In a specific embodiment, the IFN-α cytokine can induce one or more cellular responses selected from the following: inhibition of viral replication in virus-infected cells and upregulation of major histocompatibility complex I (MHC I) expression. In another specific embodiment, the IFN-α cytokine can inhibit the proliferation of tumor cells. In one embodiment, the cytokine, particularly the single-chain cytokine IFNγ, is used. In a specific embodiment, the IFN-γ cytokine can induce one or more cellular responses selected from the following: increased macrophage activity, increased MHC molecule expression, and increased NK cell activity.

[0421] In one implementation, the cytokine, particularly the single-chain cytokine IL-7, is used. In a specific implementation, IL-7 cytokine can induce the proliferation of T and / or B lymphocytes.

[0422] In one embodiment, the cytokine is IL-8. In a specific embodiment, the IL-8 cytokine can induce chemotaxis in neutrophils. In one embodiment, the cytokine, particularly the single-chain cytokine MIP-1α, is used. In a specific embodiment, the MIP-1α cytokine can induce chemotaxis in monocytes and T lymphocytes. In one embodiment, the cytokine is MIP-1β. In a specific embodiment, the MIP-1β cytokine can induce chemotaxis in monocytes and T lymphocytes. In one embodiment, the cytokine is TGF-β. In a specific embodiment, the TGF-β cytokine can induce one or more cellular responses selected from: chemotaxis in monocytes, chemotaxis in macrophages, upregulation of IL-1 expression in activated macrophages, and upregulation of IgA expression in activated B cells.

[0423] In some embodiments, the microneedles or combination therapies disclosed herein include cytokines. In these embodiments, cytokines include full-length, fragmented, or variant cytokines; cytokine receptor domains, such as cytokine receptor dimerization domains; or cytokine receptor agonists, such as antibody molecules targeting cytokine receptors (e.g., agonist antibodies).

[0424] In some embodiments, the cytokine is selected from IL-2, IL-12, IL-15, IL-18, IL-7, IL-21, or interferon-γ, or fragments or variants thereof, or combinations of any of the foregoing cytokines. The cytokine molecule may be a monomer or a dimer. In some embodiments, the cytokine molecule may also include a cytokine receptor dimerization domain.

[0425] In other embodiments, the cytokine molecule is an agonist of the cytokine receptor, such as an antibody molecule (e.g., an agonist antibody) targeting a cytokine receptor selected from IL-15Ra or IL-21R.

[0426] Other immunomodulators include, but are not limited to, cancer vaccines, such as viral cancer therapeutics and / or cancer vaccines containing tumor antigens such as neoantigens.

[0427] Surfactant formulation At least one therapeutic agent disclosed herein can be incorporated into a variety of formulations, compositions, articles, devices, and / or formulations for administration to achieve, for example, controlled and / or sustained release. More specifically, at least one therapeutic agent can be formulated into formulations, compositions, articles, devices, and / or formulations by combination with a suitable pharmaceutically acceptable carrier or diluent, and can be formulated into formulations in semi-solid, solid, or liquid form. In some embodiments, the formulations, compositions, articles, devices, and / or formulations described herein comprise silk fibroin. Exemplary formulations, compositions, articles, devices, and / or formulations include: microneedles (e.g., microneedle devices, such as microneedle patches, as described herein), implantable devices (e.g., pumps, such as subcutaneous pumps), injectable formulations, depots, gels (e.g., hydrogels), implants, and particles (e.g., microparticles and / or nanoparticles). Thus, the administration of the composition can be achieved in various ways, including intradermal, intramuscular, transdermal, subcutaneous, or intravenous administration. In addition, formulations, compositions, articles, devices and / or preparations may be formulated and / or administered to achieve controlled and / or sustained release of the therapeutic agent.

[0428] In some implementations, the therapeutic agent is administered for, for example, a duration of substantially 1, 5, 10, 15, 30, 45 minutes or at least 1, 5, 10, 15, 30, 45 minutes; a duration of 1, 2, 3, 4, 5, 10, 24 hours or at least 1, 2, 3, 4, 5, 10, 24 hours; or a duration of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days or at least 1, 2, 3, 4, 5, 6, 7... The treatment agent may be administered in timeframes of 8, 9, 10, 11, 12, 13, or 14 days; 1, 2, 3, 4, 5, 6, 7, or 8 weeks, or at least 1, 2, 3, 4, 5, 6, 7, or 8 weeks; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 months, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 months; or 1, 2, 3, 4, or 5 years or longer, or at least 1, 2, 3, 4, or 5 years or longer. In one embodiment, the therapeutic agent, such as an anticancer agent, an immunomodulator, or a combination thereof, is administered as a controlled-release or sustained-release formulation, dosage form, or device. In one embodiment, the therapeutic agent, such as an anticancer agent, an immunomodulator, or a combination thereof, is administered as a burst-release formulation, dosage form, or device. In some embodiments, the therapeutic agent is formulated for continuous delivery, such as intradermal, intramuscular, and / or intravenous continuous delivery. In some embodiments, the composition or device for controlled or sustained release of the therapeutic agent is selected from: microneedles (e.g., microneedle devices, such as microneedle patches), implantable devices (e.g., pumps, such as subcutaneous pumps), injectable formulations, reservoirs, gels (e.g., hydrogels), implants, or particles (e.g., microparticles and / or nanoparticles). In one embodiment, the therapeutic agent is in a silk fibroin-based microneedle controlled-release or extended-release dosage form or formulation (e.g., the microneedles described herein). In one embodiment, the therapeutic agent is administered via an implantable device such as a pump (e.g., a subcutaneous pump), an implantable tip of a microneedle, or a reservoir. Delivery methods can be optimized to administer and / or maintain a therapeutic dose (e.g., a standard dose) as described herein in subjects for a predetermined period of time (e.g., the following time periods or at least the following time periods: 1, 5, 10, 15, 30, 45 minutes; 1, 2, 3, 4, 5, 10, 24 hours; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days; 1, 2, 3, 4, 5, 6, 7, 8 weeks; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 months; 1, 2, 3, 4, 5 years or longer). The substantially sustained or prolonged release of the therapeutic agent can be used to prevent or treat diseases and / or conditions such as cancer for durations lasting hours, days, weeks, months, or years.

[0429] In some embodiments, this disclosure provides formulations, compositions, articles, devices, and / or preparations that are formulated and / or configured to release or sustainably release a therapeutic agent in an amount (e.g., a dose) and / or for a period of time sufficient to elicit an immune response (e.g., a cellular immune response and / or a humoral immune response) against an antigen (e.g., a tumor-specific antigen, such as a neoantigen) in a subject.

[0430] In some embodiments, the formulations, compositions, articles, devices and / or preparations of this disclosure may be formulated and / or configured for controlled or sustained release of at least one therapeutic agent in an amount (e.g., dose) and / or for a period of time sufficient to induce immunity (e.g., cancer immunity) in a subject.

[0431] Therapeutic agents are essentially continuously or prolongedly released or formulated for the prevention or treatment of diseases or conditions such as cancer for a period of time lasting hours, days, weeks, months or years.

[0432] In some embodiments, the therapeutic agents described herein may be added to a silk fibroin solution, for example, prior to the formation of the silk fibroin microneedles or microneedle devices described herein. In embodiments, the silk fibroin solution may be mixed with the therapeutic agents and then used in the manufacture of implantable microneedle tips, for example by processes such as filling and / or casting, drying, and / or annealing to produce microneedles having any of the desired material properties as described herein.

[0433] Unbound by theory, the ratio of silk fibroin to therapeutic agent in the silk fibroin tip of the microneedle (e.g., an implantable tip) will affect its release. In some embodiments, an increased silk concentration in the tip favors slower release and / or greater retention of therapeutic agent within the tip. Any concentration of silk can be used, as long as it allows for printing and has sufficient mechanical strength to pierce the skin.

[0434] In some embodiments, silk fibroin may be used at a concentration of about 1% w / v to about 10% w / v (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% w / v) in the manufacture of microneedles or components thereof as described herein. In some embodiments, silk fibroin may be used at a concentration of about 1% w / v to about 30% w / v (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30% w / v) in the manufacture of microneedles or components thereof as described herein.

[0435] In some embodiments, in the manufacture of microneedles or components thereof as described herein, silk fibroin may be used in amounts from about 0.5 μg to about 500 μg. In some embodiments, in the manufacture of microneedles or components thereof as described herein, silk fibroin may be used in amounts from about 0.5 µg to about 5 µg, or about 1 µg to about 10 µg, or about 5 µg to about 15 µg, or about 10 µg to about 20 µg, or about 15 µg to about 25 µg, or about 20 µg to about 30 µg, or about 25 µg to about 35 µg, or about 30 µg to about 40 µg, or about 35 µg to about 45 µg, or about 40 µg to about 50 µg, or about 45 µg to about 55 µg, or about 50 µg to about 60 µg, or about 55 µg to about 65 µg, or about 60 µg to about 70 µg, or about 65 µg... The amount of silk fibroin used is from about 75 µg to about 70 µg to about 80 µg, or about 75 µg to about 85 µg, or about 80 µg to about 90 µg, or about 85 µg to about 95 µg, or about 90 µg to about 100 µg, or about 95 µg to about 150 µg, or about 125 µg to about 175 µg, or about 150 µg to about 200 µg, or about 225 µg to about 275 µg, or about 250 µg to about 300 µg, or about 325 µg to about 375 µg, or about 350 µg to about 400 µg, or about 425 µg to about 475 µg, or about 450 µg to about 500 µg. In some embodiments, in the manufacture of microneedles or components thereof as described herein, silk fibroin may be used in amounts of at least about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, or 500 µg of silk fibroin. In some embodiments, in the manufacture of microneedles or components thereof as described herein, silk fibroin may be used in amounts of up to about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, or 500 µg of silk fibroin.In some embodiments, in the manufacture of microneedles or components thereof as described herein, silk fibroin may be used in amounts of about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, or 500 µg of silk fibroin. In some embodiments, in the manufacture of microneedles or components thereof as described herein, silk fibroin may be used in amounts from about 2.42 µg to 242 µg.

[0436] In some embodiments, in the manufacture of microneedles or components thereof as described herein, silk fibroin may be used in an amount of about 1% to about 75%, about 1% to about 5%, about 10% to about 60%, about 15% to about 50%, or about 20% to about 40% by weight. In some embodiments, in the manufacture of microneedles or components thereof as described herein, silk fibroin may be used in an amount of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75% by weight. In some embodiments, in the manufacture of microneedles or components thereof as described herein, silk fibroin may be used in an amount of up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75% by weight.

[0437] Exemplary excipients Additionally, the formulations, compositions, articles, devices, and / or formulations may be formulated with commonly used excipients, diluents, or carriers for administration via intradermal, intramuscular, transdermal, subcutaneous, or intravenous routes. In some embodiments, the formulations, compositions, articles, devices, and / or formulations may be administered, for example, transdermally, and may be formulated as controlled-release or sustained-release dosage forms, etc. The formulations, compositions, articles, devices, and / or formulations described herein may be administered alone, in combination with each other, or they may be used in combination with other known therapeutic agents.

[0438] Formulations applicable to this disclosure can be found in Remington's Pharmaceutical Sciences (1985). Furthermore, for a review of drug delivery methods, see Langer (1990). Science 249:1527-1533. The formulations, compositions, articles, devices, and / or formulations described herein can be manufactured in ways known to those skilled in the art, such as by mixing, dissolving, granulating, forming sugar-coated pellets, spinning into fine powder, emulsifying, encapsulating, embedding, or lyophilizing processes. The methods and excipients described below are exemplary only and are not intended to be limiting.

[0439] The silk fibroin formulations used in the manufacture of the microneedles described herein may include excipients. In embodiments, excipients may be included for the purpose of: improving the stability of the introduced therapeutic agent, such as an anticancer agent, immunomodulator, or a combination thereof; increasing the porosity of the silk matrix and the diffusion of the therapeutic agent, such as an anticancer agent, immunomodulator, or a combination thereof, from the formulation, composition, article, device, formulation, and / or microneedle (e.g., microneedle tip); and / or increasing the crystallinity / β-sheet content of the silk matrix to make the silk material insoluble.

[0440] Exemplary excipients include, but are not limited to, sugars or sugar alcohols (e.g., sucrose, trehalose, sorbitol, mannitol, or combinations thereof), divalent cations (e.g., Ca2+), and sugars or sugar alcohols (e.g., sucrose, trehalose, sorbitol, mannitol, or combinations thereof), and divalent cations (e.g., Ca2+). 2+ Mg 2+ Mn 2+ and Cu 2+ Excipients include surfactants (e.g., octylphenol ethoxylates (e.g., Triton-X), polysorbates, poloxamers, and / or polyethoxylated alcohols), polyols (e.g., glycerol), glycols (e.g., propylene glycol, PEG), and / or buffers. In some embodiments, the concentration of excipients can be used to alter the porosity of the matrix; for example, sucrose is the most commonly used excipient for this purpose. Excipients can also be added to promote the self-assembly of filaments into ordered β-sheet secondary structures, and such excipients typically participate in hydrogen bonding or charge interactions with the filaments to achieve this effect. Non-limiting examples of excipients that can be used to promote the self-assembly of filaments into ordered β-sheet secondary structures include monosodium glutamate (e.g., L-glutamate), lysine, sugar alcohols (e.g., sorbitol and / or glycerol), and solvents (e.g., DMSO, methanol, and / or ethanol).

[0441] In some embodiments, the sugar or sugar alcohol is sucrose, present in amounts of less than 70% (w / v), less than 60% (w / v), less than 50% (w / v), less than 40% (w / v), less than 30% (w / v), less than 20% (w / v), less than 10% (w / v), less than 9% (w / v), less than 8% (w / v), less than 7% (w / v), less than 6% (w / v), or 5% (w / v) or less, for example, just before drying.

[0442] In some embodiments, the sugar or sugar alcohol is sucrose, present in amounts of about 1% (w / v) to about 10% (w / v), about 2% (w / v) to about 8% (w / v), about 2.2% (w / v) to about 6% (w / v), about 2.4% (w / v) to about 5.5% (w / v), about 2.5% to about 5%, or about 2.4% (w / v), about 2.5%, or about 5% (w / v), for example, just before drying.

[0443] In some embodiments, the sugar or sugar alcohol is trehalose, present in amounts of about 1% (w / v) to about 10% (w / v), about 2% (w / v) to about 8% (w / v), about 2.2% (w / v) to about 6% (w / v), about 2.4% (w / v) to about 5.5% (w / v), about 2.5% to about 5%, or about 2.4% (w / v), about 2.5%, or about 5% (w / v), for example, just before drying.

[0444] In some embodiments, the sugar or sugar alcohol is sorbitol, present in amounts of about 1% (w / v) to about 10% (w / v), about 2% (w / v) to about 8% (w / v), about 2.2% (w / v) to about 6% (w / v), about 2.4% (w / v) to about 5.5% (w / v), about 2.5% to about 5%, or about 2.4% (w / v), about 2.5%, or about 5% (w / v), for example, just before drying.

[0445] In some embodiments, the sugar or sugar alcohol is glycerol, present in amounts of about 1% (w / v) to about 10% (w / v), about 2% (w / v) to about 8% (w / v), about 2.2% (w / v) to about 6% (w / v), about 2.4% (w / v) to about 5.5% (w / v), about 2.5% to about 5%, or about 2.4% (w / v), about 2.5%, or about 5% (w / v), for example, just before drying.

[0446] In some embodiments, the surfactant (e.g., octylphenol ethoxylate (e.g., Triton-X), polysorbate, poloxamer and / or polyethoxylated alcohol) is present in amounts of about 0.005% (w / v) to about 1% (w / v), about 1% (w / v) to about 10% (w / v), about 2% (w / v) to about 8% (w / v), about 2.2% (w / v) to about 6% (w / v), about 2.4% (w / v) to about 5.5% (w / v), about 2.5% to about 5%, or about 2.4% (w / v), about 2.5%, or about 5% (w / v), for example, just before drying.

[0447] In some embodiments, the polyol (e.g., glycerol) is present in amounts of about 1% (w / v) to about 10% (w / v), about 2% (w / v) to about 8% (w / v), about 2.2% (w / v) to about 6% (w / v), about 2.4% (w / v) to about 5.5% (w / v), about 2.5% to about 5%, or about 2.4% (w / v), about 2.5%, or about 5% (w / v), for example, just before drying.

[0448] In some embodiments, the glycol (e.g., propylene glycol, such as PEG) is present in amounts of about 1% (w / v) to about 10% (w / v), about 2% (w / v) to about 8% (w / v), about 2.2% (w / v) to about 6% (w / v), about 2.4% (w / v) to about 5.5% (w / v), about 2.5% to about 5%, or about 2.4% (w / v), about 2.5%, or about 5% (w / v), for example, just before drying.

[0449] In some embodiments, the therapeutic formulation further comprises a divalent cation. In some embodiments, the divalent cation is selected from Ca... 2+ Mg 2+ Mn 2+ and Cu 2+ In some embodiments, for example, prior to drying, the divalent cation is present in the formulation at an amount from 0.1 mM to 100 mM. In some embodiments, for example, prior to drying, the divalent cation is present at 10 per standard dose of the therapeutic agent (e.g., an anticancer agent, an immunomodulator, a viral immunogen, or a combination thereof). -7 Up to 10 -4 The molar amount exists in the formulation. In some embodiments, the divalent cation is in 10 molar amounts before drying. -10 Up to 2×10 -3 The molar amount exists in the formulation.

[0450] In some implementations, the therapeutic agent also comprises poly(lactic-co-glycolic acid) (PGLA).

[0451] In some embodiments, such as just before drying, the therapeutic formulation also includes a buffer. In some embodiments, the buffer has a buffering capacity of pH 3 to pH 8, pH 4 to pH 7.5, or pH 5 to pH 7. In some embodiments, the buffer is selected from PBS, HEPES, and CP buffer. In some embodiments, such as just before drying, the buffer is present in the formulation at an amount of 0.1 mM to 100 mM. In some embodiments, the buffer is present at 10 mM per standard dose of the therapeutic agent (e.g., an anticancer agent, an immunomodulator, a viral immunogen, or a combination thereof). -7 Up to 10 -4 The amount is present in moles. In some implementations, the buffer is in 10... -10 Up to 2×10 -3 The amount of moles exists.

[0452] Additionally, the therapeutic agent can be formulated as a reservoir, gel, or hydrogel formulation. Such long-acting formulations can be administered via implantation (e.g., subcutaneous or intramuscular) or intramuscular injection. Thus, for example, the therapeutic agent can be formulated with a suitable polymer or hydrophobic material (e.g., as an emulsion in an acceptable oil) or with an ion exchange resin, or as a slightly soluble derivative, such as a slightly soluble salt.

[0453] In one embodiment, the therapeutic agent is administered via an implantable infusion device such as a pump (e.g., a subcutaneous pump), implant, or reservoir. An implantable infusion device typically includes a housing that houses a fluid reservoir, which can be transdermally filled by a subcutaneous injection needle through a filling port septum. The drug reservoir is typically connected to the device's outlet port via an internal flow path to deliver the fluid to the patient's body site via a catheter. A typical infusion device also includes a controller and fluid delivery mechanisms, such as a pump or valve, to move the fluid from the reservoir through the internal flow path to the device's outlet port.

[0454] In some embodiments, the therapeutic agent may be packaged and / or formulated as particles, such as micron-sized particles and / or nanoparticles. Typically, nanoparticles have diameters of 10, 15, 20, 25, 30, 35, 45, 50, 75, 100, 150, or 200 nm, or from 200 nm to 1,000 nm, for example, 10, 15, 20, 25, 30, 35, 45, 50, 75, 100, 150, or 200, or 20, 30, or 50-400 nm. Smaller particles tend to be removed from the system more quickly. The therapeutic agent (including those described herein) may be embedded within or coupled to (e.g., covalently coupled to) or otherwise adhered to the nanoparticle.

[0455] Lipid- or oil-based nanoparticles, such as liposomes and solid lipid nanoparticles, can be used to deliver therapeutic agents such as anticancer agents, immunomodulators, or combinations thereof, as described herein. Solid lipid nanoparticles for delivering therapeutic agents are described (see, for example, Serpe et al., (2004) Eur. J. Pharm. Bioparm. 58:673-680, and Lu et al. (2006 Eur. J. Pharm. Sci. 28: 86-95). Polymer-based nanoparticles (e.g., PLGA-based nanoparticles) can be used to deliver the agents described herein. These tend to rely on a biodegradable backbone in which the therapeutic agent is intercalated (with or without covalent bonds to the polymer) within the polymer matrix. PLGA is widely used in polymer nanoparticles (see, for example, Hu et al., (2009) J. Control. Release 134:55-61; Cheng et al., (2007) Biomaterials 28:869-876; and Chan et al., (2009) Biomaterials 30:1627-1634). PEGylated PLGA-based nanoparticles can also be used to deliver therapeutic agents (see, for example, Danhhier et al., (2009) J. Control. Release 133:11-17; Gryparis et al., (2007) Eur. J. Pharm. Biopharm. 67:1-8). Metal-based nanoparticles (e.g., gold-based nanoparticles) can also be used to deliver therapeutic agents. Protein-based nanoparticles (e.g., albumin-based nanoparticles) can be used to deliver the therapeutic agents described herein. In some embodiments, the therapeutic agent may be bound to human albumin nanoparticles.

[0456] A wide range of nanoparticles are known in the art. Exemplary methods include those described in WO2010 / 005726, WO2010 / 005723, WO2010 / 005721, WO2008 / 121949, WO2010 / 075072, WO2010 / 068866, WO2010 / 005740, WO2006 / 014626, US 7,820,788, and US 7,780,984, the contents of which are incorporated herein by reference in their entirety.

[0457] dose The methods described herein may be used in any dose (e.g., standard dose and / or fractional dose) of a therapeutic agent that, when administered via microneedles of this disclosure, is capable of evoking a therapeutic response (e.g., an anticancer response, or an immune response) in a subject.

[0458] Without being bound by theory, the total dose of therapeutic agent to be applied via the microneedles described herein (e.g., a standard dose) may be distributed among multiple microneedles (e.g., within a patch) such that a single microneedle tip may contain less than about 1% of the total dose (e.g., in an array containing about 121 microneedles), or at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% or more of the total dose.

[0459] In some embodiments, the dose of therapeutic agent loaded into the microneedle patch can be controlled by the concentration of the therapeutic agent in the formulation solution forming the needle tips, the volume of solution dispensed into each needle tip, and the total number of needles. In some embodiments, the former two are more convenient means of changing the dose. The dose released into the subject is related to deployment efficiency (the portion of the needle tip remaining in the biological barrier, such as the skin, after the patch is removed) and the release profile over time and the residence time of the tip in the subject. Since the skin is constantly shed from the epidermis, deeper deployment within the skin is associated with a longer residence time. Therefore, it is desirable to maximize the penetration depth of the needle tips (reaching the limit defined by the depth of pain receptors within the skin, for example, a depth of about 100 μm to about 600 μm), and it is also desirable to spatially concentrate the antigen toward the needle tips.

[0460] The formulations, compositions, articles, devices, and / or formulations described herein (including silk fibroin tip formulations) are designed to release (e.g., sustained release) a therapeutic agent not only for the duration the tip remains in the dermis, but also to maintain the stability of the therapeutic agent during this period (e.g., at least about 1-2 weeks). In some embodiments, it is anticipated that approximately 95-100% of the total dose amount introduced into the formulations, compositions, articles, devices, formulations, and / or microneedles described herein may be available for delivery to, for example, a subject, such as to the subject's tissues, such as skin, tumors, mucous membranes, organ tissues, buccal cavities, tissues, or cell membranes. Not bound by theory, microneedles are successfully deployed into the skin for at least about 50% and up to 100% (e.g., at least about 50%, 60%, 70%, 80%, 90%, or more (e.g., 100%) of the total number of microneedles in the array after application) to achieve controlled or sustained release of a therapeutic agent such as an anticancer agent, an immunomodulator, or a combination thereof. In some implementations, a portion of the antigen may not be released from the filament tip during the duration of deployment.

[0461] Methods for manufacturing microneedles Figure 1The diagram illustrates an exemplary method for manufacturing the microneedles of this disclosure. Machine vision-guided dispensing of precise nanoliter (nL) volumes of silk fibroin solution into individual needle cavities allows for the introduction of different doses and formulations into the releasable tip of a microneedle device (e.g., a microneedle array or patch). An exemplary microneedle device (e.g., a microneedle array or patch) comprises an 11×11 array of cones. It should be understood that microneedle devices may include needle cavities generated in arrays with different numbers and orientations to achieve desired results.

[0462] Mold production In some implementations, a mold is used in the fabrication of the microneedle device. As will be discussed in more detail below, sterile molds are used to produce microneedle devices having an array of releasable tips embodying a therapeutic agent-filament formulation (e.g., an anticancer agent-filament formulation, an immunomodulatory agent-filament formulation, an antigen-filament formulation, or a combination thereof).

[0463] For example, a male mold with a microneedle array of desired geometry can be used to cast silicone (DOW CorningSylgard® 184) resin. Once the silicone has cured, it can be removed from the mold. The mold can then be reused for mass silicone casting. Defects in the silicone mold can be inspected throughout the manufacturing process (e.g., between castings). If necessary, the silicone mold can be sterilized, for example, by autoclaving. In one embodiment, the mold includes a mold body having an array of needle cavities formed within the mold body.

[0464] In some implementations, other types of silicone and / or other materials and processes may be used to manufacture the mold. For example, liquid silicone injection molding and thermoplastic elastomer injection molding may be used. It is not to be bound by theory, but it is understood that a key requirement is that the mold material is soft and flexible (e.g., having a Shore hardness of approximately 50 A) and has low adhesion to the filaments and other materials used in the construction of the patch.

[0465] Tip filler The tip formulation, consisting of silk fibroin, therapeutic agents (e.g., anticancer agents, immunomodulators, antigens, or combinations thereof) and possibly other excipients in an aqueous solution, is dispensed via nanoliter printing into each needle cavity of a die. Currently, this is accomplished at the laboratory scale using machine vision-guided automated dispensing systems, such as the Biojet Elite™ AD3400 dispensing system manufactured by BioDot, but systems with similar capabilities manufactured by other suppliers may also be used. In some embodiments, the working volume of the dispenser used for tip filling (e.g., a BioDot™ dispenser) is enclosed and maintained at elevated relative humidity (RH), such as 60% or higher, to, for example, slow down the drying of the formulation and / or prevent the accumulation of dried solids on the dispensing nozzle. In one embodiment, the working volume of the BioDot™ dispenser is enclosed and maintained at 60% relative humidity (RH) to slow down the drying of the formulation and / or prevent the accumulation of dried solids on the dispensing nozzle.

[0466] The mold is placed within a fixture that constrains its position on the BioDot™ dispenser's processing platform. The machine uses a camera to image each mold, and machine vision algorithms determine the precise location and orientation of the needle cavity array within each mold. This location guides subsequent dispensing steps. A stereomicroscope is used to inspect the filled mold for filling defects, such as misaligned dispensing or large air bubbles in the liquid.

[0467] Primary drying In some embodiments, the filled mold is set aside to dry, for example, within a housing that maintains the desired ambient humidity. In one embodiment, the filled mold is set aside to dry within a machine housing for approximately 7 minutes. In some embodiments, the solubility of silk fibroin can be adjusted by controlling the drying time. It is not desirable to be bound by theory; during drying, the silk structure may transform into more β-sheets and become less soluble (e.g., insoluble), and this effect can be increased by drying more slowly and / or by incubating at increased humidity (e.g., from approximately 10% to approximately 100% RH).

[0468] After drying, the above dispensing process can be repeated. In some embodiments, the mold is moved to a chamber with approximately saturated humidity and incubated overnight to slowly dry the tip. During this time, the filament structure can transform into more β-folds and become less soluble (e.g., insoluble) (annealing).

[0469] Secondary drying In some implementations, the mold is moved into a chamber where the humidity is controlled at a relative humidity (RH) of about 10% to about 25% and the ambient room temperature and left overnight (about 14 hours) to complete the drying process. This is a “secondary” drying step.

[0470] Water annealing In some implementations, the mold (e.g., a mold containing dried silk fibroin tips) is transferred to a vacuum desiccator also containing approximately 500 mL of deionized water (DIW). The desiccator is shut off and a vacuum is applied in the laboratory using the main vacuum line for approximately 5 minutes. After 5 minutes, the desiccator's outlet valve is closed and the mold is placed in an incubator maintained at 37°C for four hours. After four hours, the desiccator is vented and the mold is transferred back to a 25% RH chamber at ambient room temperature.

[0471] Drying after annealing Before subsequent steps, the mold can be kept at approximately 10% to approximately 25% RH for at least four hours or at most overnight.

[0472] Base layer filling A soluble base layer can be formed by filling the mold with the base solution described herein. In some embodiments, the base solution contains 40% w / v hydrolyzed gelatin and 10% w / v sucrose in the DIW. In some embodiments, the base solution contains 30% dextran 70 kDa, 10% sucrose, 1% glycerol, and 0.01% Triton-X100. The base layer can be filled in any suitable manner. For example, firstly, a volume of base solution suitable for the mold (e.g., 150 μL) can be evenly spread over the mold using a pipette. The mold can then be centrifuged (e.g., at 3900 rpm for up to about 2 minutes). The mold can be checked, and if any needle cavities remain unfilled, the filling and centrifugation process can be repeated. The mold can be further “filled” with a suitable amount of base solution (e.g., 50 μL of base solution). In some embodiments, centrifugal filling can be used. In some embodiments, the base is filled in the same manner as the tip by dispensing a visually guided droplet into the mold cavity.

[0473] In some embodiments, more than one base layer may be applied. For example, a first base layer may be formed according to the method described above. Then, for example, after drying the first base layer, the process may be repeated to add one or more additional base layers. The one or more additional base layers may be applied using the same base layer solution used for the first base layer, or one or more different base layer solutions may be used as needed (e.g., one or more base layer solutions described herein). In some embodiments, the base solution is a molten liquid or slurry. In some embodiments, a chemical reaction (e.g., after filling) is used to solidify the base layer.

[0474] Base drying The filled mold can be transferred back to a chamber at approximately 10% to approximately 25% RH and dried for at least overnight and up to 3 days.

[0475] Backing application Patches used to generate the release of therapeutic agents (e.g., controlled release or sustained release) and / or improve immunogenicity (see examples, for example) have a paper backing layer; however, subsequent developments have shown that adhesive plastic tapes can have excellent properties as a backing layer.

[0476] In some embodiments, the paper backing process is as follows: 10-30 μL of DIW is spread on the surface using a pipette to rewet the dried substrate portion. Whatman 903 paper is punched into a circle with a diameter of 12 mm. The paper circle is gently pressed into the wet surface of the substrate. The wet substrate is partially immersed in the paper. The mold with the backing is transferred back to a chamber at 25% RH to dry for at least 4 hours until use. In some embodiments, the backing (e.g., a backing containing an adhesive) is cured by light irradiation.

[0477] Adhesive tape process Cut adhesive-backed polyester tape (e.g., 3M® magic™ tape) into pieces approximately 12 mm wide and 25 mm long. Align one end of the tape with the adhesive and gently press it onto the surface of the base layer. Fold the free end of the tape over itself to form a non-adhesive "handle".

[0478] Demolding Remove the patch from the mold before use. Gently bend the flexible mold away from the stiffer patch and remove the patch from the mold. Inspect the patch for defects such as missing or broken pins.

[0479] Package In the study above, the patch was used shortly after demolding and was not packaged. If long-term storage is required, the assembled patch can be packaged with a desiccant in a container with low water vapor permeability (e.g., a glass vial or thermoformed plastic tray made of a low MVTR material and a foil-backed heat-sealed cap) to maintain a relative humidity of about 0% to about 50% (e.g., about 0% to 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, or about 40% to 50%, such as about 25%) within the package.

[0480] Therapeutic applications In one aspect, this disclosure provides methods for delivering (e.g., administering) an effective amount of a therapeutic agent, such as an anticancer agent, an immunomodulator, or a combination thereof, across biological barriers (e.g., skin layer, cell membrane, mucous surface, oral cavity, skin lesion, tumor, or buccal cavity).

[0481] In one aspect, this disclosure provides methods for treating, preventing, and / or improving diseases and / or conditions in a subject, such as cancer and / or skin conditions. In some embodiments, delivery is intratumoral. However, in some embodiments, microneedles may be applied to sites adjacent to the tumor (e.g., peritumoral delivery). In some embodiments, microneedle application is used as a first-line therapy, such as for unresectable tumors. In some embodiments, microneedle application is used as a neoadjuvant therapy (e.g., prior to primary treatment such as surgery to remove the tumor) to shrink the tumor. In some embodiments, microneedle application is used as an adjuvant therapy (e.g., after tumor resection) to reduce the risk of cancer recurrence.

[0482] In one aspect, this disclosure relates to a method of treating a subject's cancer. The method includes administering a microneedle of this disclosure to the subject to treat the subject's cancer. Exemplary cancers that can be treated with the microneedles of this disclosure are known in the art and are disclosed herein.

[0483] In one aspect, this disclosure relates to a method of treating a skin condition in a subject. The method includes administering microneedles of this disclosure to the subject to treat the subject's cancer. Exemplary skin conditions that can be treated with the microneedles of this disclosure are known in the art and are disclosed herein.

[0484] In one aspect, this disclosure relates to methods for inducing anticancer and / or immune responses (e.g., local and / or systemic immune responses) in subjects with such needs. In some embodiments, the anticancer and / or immune responses (e.g., local and / or systemic immune responses) in the subject include: reduction in tumor volume or cancer volume, reduction in the number of tumor cells or cancer cells, reduction in the number of metastases, increase in life expectancy, reduction in tumor cell proliferation or cancer cell proliferation, reduction in tumor cell survival or cancer cell survival, prevention of recurrence, and / or improvement in various physiological symptoms associated with the cancer condition.

[0485] In some embodiments, this disclosure relates to methods for inducing anticancer and / or immune responses (e.g., local and / or systemic immune responses) in subjects in need of such responses, resulting in a reduction in tumor volume or cancer volume in the subject, for example, by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% or more compared to baseline.

[0486] In some embodiments, this disclosure relates to methods for inducing anticancer and / or immune responses (e.g., local and / or systemic immune responses) in subjects in need of such responses, resulting in a reduction in the number of tumor cells or cancer cells in the subject, for example, by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% or more compared to a baseline value.

[0487] In some embodiments, this disclosure relates to a method of inducing an anticancer response and / or an immune response (e.g., a local and / or systemic immune response) in a subject in need of such response, which results in a reduction in the number of metastases in the subject, for example, by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% or more compared to a baseline value.

[0488] In some embodiments, this disclosure relates to methods for inducing anticancer and / or immune responses (e.g., local and / or systemic immune responses) in subjects in need of such responses, which extend the lifespan of the subjects by at least about 15, 30, 60, 90, 120, 180, or 360 days.

[0489] In some embodiments, this disclosure relates to a method of inducing an anticancer response and / or an immune response (e.g., a local and / or systemic immune response) in a subject in need of such an response, which results in a reduction of tumor cell proliferation or cancer cell proliferation, for example, by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% or more compared to a baseline value.

[0490] In some embodiments, this disclosure relates to methods for inducing anticancer and / or immune responses (e.g., local and / or systemic immune responses) in subjects in need of such responses, resulting in a reduction of tumor cell survival or cancer cell survival, for example, by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% or more compared to baseline values.

[0491] In one aspect, this disclosure provides methods for treating diseases associated with the expression of tumor-specific antigens (e.g., neoantigens).

[0492] In one aspect, this disclosure relates to methods for inhibiting the growth of tumors and / or skin lesions (e.g., tumors and / or skin lesions expressing tumor-specific antigens (e.g., neoantigens)). In some embodiments, the tumor to be treated may be associated with cancer-specific or tumor-specific antigens that may not be present in normal cells. However, it is not desirable to be bound by the theory that neoantigens may be poorly or inefficiently presented to the subject's immune system, for example, due to immunosuppressive tumor microenvironment (TME). In some embodiments, the methods described herein can improve the presentation of neoantigens to the subject's immune system and promote robust and long-lasting immune responses (e.g., anti-cancer responses, such as cancer immunity) in the subject. In some embodiments, the methods described herein can result in tumor ablation at or near the initial microneedle application site and also result in tumor ablation at distant sites within the subject, such as anywhere cancer cells expressing neoantigens are present.

[0493] This disclosure provides fibroin-based microneedles and fibroin-based microneedle devices for treating cancer (e.g., metastatic cancer) and / or skin conditions (e.g., dermatitis) and / or inducing an immune response to cancer (e.g., metastatic cancer) and / or skin conditions (e.g., dermatitis). In some embodiments, the methods disclosed herein include contacting (e.g., administering) a microneedle device or multiple microneedles containing an anticancer agent, an immunomodulatory agent, or a combination thereof with a site of cancer (e.g., metastatic tumor) or lesion (e.g., skin lesion) in a subject, thereby generating one or more of the following: (i) lysing cancer cells, such as tumor cells, to release cancer-associated antigens (e.g., neoantigens) and / or exposing cancer-associated antigens (e.g., neoantigens) to the subject's immune system; (ii) displaying cancer-associated antigens (e.g., neoantigens) complexed with the major histocompatibility complex (MHC) on their surface by antigen-presenting cells (APCs); (iii) recognizing the displayed cancer-associated antigens (e.g., neoantigens) by immune effector cells (e.g., T cells and / or NK cells); (iv) activating and / or expanding immune effector cells, such as T cells and / or NK cells, that are specific for the cancer-associated antigens (e.g., neoantigens) displayed in the subject; and (v) Enhance (e.g., stimulate or upregulate) the immune response of immune effector cells such as T cells and / or NK cells, which promote the killing of target cells expressing cancer-associated antigens (e.g., neoantigens) in the subject and / or inhibit the growth or proliferation of said target cells.

[0494] In one aspect, this disclosure relates to methods of releasing tumor-specific antigens (e.g., neoantigens) and / or exposing tumor-specific antigens (e.g., neoantigens) to the immune system of a subject to elicit an anticancer response and / or an immune response.

[0495] In one aspect, this disclosure relates to a method for inducing cancer immunity in a subject, optionally wherein said cancer immunity is targeted at a tumor-specific antigen (e.g., a neoantigen).

[0496] In some embodiments of any of the methods described herein, the application of microneedles causes immune cells to activate in response to tumor-specific antigens (e.g., neoantigens) exposed and / or released from the tumor due to treatment with microneedles (e.g., microneedles containing anticancer agents and immunomodulators and / or combinations thereof). Furthermore, the activated immune cells target cancer cells expressing tumor-specific antigens (e.g., neoantigens), thereby inhibiting cancer growth and / or proliferation. Optionally, the targeted cancer cells are located at or near the site of microneedle application. Optionally, the targeted cancer cells are located at a distant site.

[0497] In one aspect, this disclosure relates to a method for inducing an anticancer response in a subject. In some embodiments, the method includes administering microneedles of this disclosure to the subject to induce an immune response, for example, against a disease associated with the expression of a tumor-specific antigen (e.g., a neoantigen). In any aspect of the methods described herein, immunity (e.g., cancer immunity) persists in the subject for a period of time after the administration of the microneedles. For example, immunity (e.g., cancer immunity) may persist in the subject for about one week, about two weeks, about three weeks, about one month, about two months, about three months, about four months, about five months, about six months, about seven months, about eight months, about nine months, about ten months, about eleven months, about twelve months, about thirteen months, about fourteen months, about fifteen months, about sixteen months, about seventeen months, about eighteen months, about nineteen months, about twenty months, about twenty-one months, about twenty-two months, about twenty-three months, about two years, about three years, about four years, or about five years after the administration of the microneedles.

[0498] In one aspect, this article provides a method for delivering (e.g., administering) an effective amount of a therapeutic agent, such as an anticancer agent, an immunomodulatory agent, or a combination thereof, to the tumor site after tumor resection to, for example, induce an immune response to the tumor and / or ablate any cancer cells remaining after resection.

[0499] In one aspect, this article provides a method for delivering (e.g., administering) an effective amount of a therapeutic agent, such as an anticancer agent, an immunomodulatory agent, or a combination thereof, to a tumor site prior to tumor resection to, for example, induce an immune response to the tumor and / or ablate any cancer cells remaining after resection.

[0500] Such methods may include providing microneedles containing therapeutic agents such as anticancer agents, immunomodulators, or combinations thereof as described herein. For example, such methods may include providing at least one microneedle or at least one microneedle device described herein, wherein the microneedle or microneedle device comprises a silk fibroin-based tip having a therapeutic agent such as an anticancer agent, immunomodulator, or combination thereof; penetrating the microneedle or microneedle device into a biological barrier (e.g., skin, for example, a tumor); and allowing an effective amount of the therapeutic agent to be released from the silk fibroin tip over a period of time, for example, at least about 1 day (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days or more, for example, about 4 days to about 14 days, for example, about 1-2 weeks, about 1-3 weeks, or about 1-4 weeks, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 months, for example, 1, 2, 3, 4, 5 years or longer).

[0501] Combination therapy The microneedles disclosed in this article can be used in combination with a second therapeutic agent or procedure.

[0502] In some embodiments, the microneedles and the second therapeutic agent or procedure disclosed herein are administered / performed after the subject has been diagnosed with cancer, for example, before the cancer has been eliminated from the subject. In other embodiments, the microneedles are administered / performed simultaneously or concurrently with the second therapeutic agent or procedure. For example, the delivery of one treatment may be in progress while the delivery of the second treatment has begun, for example, there is overlap in the administration of treatments. In other embodiments, the microneedles are administered / performed sequentially with the second therapeutic agent or procedure. For example, the delivery of one treatment may be stopped before the delivery of another treatment begins.

[0503] In the implementation scheme, the combination therapy can produce a more effective treatment than a monotherapy using either agent alone. In the implementation scheme, the combination of the first and second treatments is more effective than the first or second treatment alone (e.g., resulting in greater symptom relief and / or a greater reduction in cancer cells). In the implementation scheme, the combination therapy allows for the use of lower doses of the first or second treatment compared to the doses typically required to achieve a similar effect when administered as monotherapy. In the implementation scheme, the combination therapy has a partial additive effect, a complete additive effect, or a greater than additive effect.

[0504] In one implementation, microneedle molecules are administered in combination with a therapy such as a cancer therapy (e.g., one or more of anticancer agents, immunotherapy, photodynamic therapy (PDT), surgery, and / or radiation). The terms “chemotherapeutic agent,” “chemotherapy agent,” and “anticancer agent” are used interchangeably herein. The administration of microneedles to the said therapy, such as the cancer therapy, may be sequential (with or without overlap) or simultaneous. During the course of a therapy (e.g., cancer therapy), the administration of microneedles may be continuous or intermittent. Some of the therapies described herein can be used to treat cancerous and non-cancerous conditions. For example, the methods and compositions described herein can enhance the efficacy of therapeutic agents in both cancerous and non-cancerous conditions.

[0505] Patient selection In any method of treating a subject or in some embodiments of the compositions used for the purposes disclosed herein, the subject... The test subject has a medical condition, such as cancer.

[0506] As used herein, “cancer” can encompass all types of carcinogenic processes and / or cancerous growth. In implementation, cancer includes primary tumors as well as metastatic tissue or malignantly transformed cells, tissues, or organs. In implementation, cancer encompasses all histopathological and stage-specific aspects of cancer, such as aggressive / severe stages. In implementation, cancer includes recurrent and / or drug-resistant cancers. The terms “cancer” and “tumor” are used interchangeably. For example, both terms cover solid tumors and liquid tumors. As used herein, the terms “cancer” or “tumor” include both pre-existing and malignant cancers and tumors. This article describes examples of various cancers, including but not limited to: anal cancer; basal cell carcinoma; bladder cancer; bone cancer; brain tumors; breast cancer; cervical cancer; colon and rectal cancer; endometrial cancer; esophageal cancer; gastrointestinal cancer (e.g., gastrointestinal stromal tumors); gestational trophoblastic disease; head and neck cancer; Hodgkin lymphoma; Kaposi's sarcoma; kidney (renal cell) carcinoma; leukemia; liver cancer; lung cancer; malignant mesothelioma; melanoma; Merkel cell carcinoma; multicentric Castleman's disease; multiple myeloma and other plasma cell tumors; myeloproliferative neoplasms; neuroblastoma; non-Hodgkin lymphoma; ovarian cancer, fallopian tube cancer, or primary peritoneal cancer; pancreatic cancer; penile cancer; pheochromocytoma and paraganglioma; prostate cancer; retinoblastoma; rhabdomyosarcoma; skin cancer; squamous cell carcinoma; soft tissue sarcoma; solid tumors anywhere in the body; stomach (gastric) cancer; testicular cancer; thyroid cancer; vaginal cancer; vulvar cancer; and Wilms' tumor and other childhood kidney cancers, etc.

[0507] In some embodiments, the cancer is melanoma. In some embodiments, the cancer is basal cell carcinoma. In some embodiments, the cancer is squamous cell carcinoma. In some embodiments, the cancer is Merkel cell carcinoma. In some embodiments, the cancer is breast cancer.

[0508] In any method of treating a subject or in some embodiments of the compositions used for the purposes disclosed herein, the subject has a skin condition. Examples of various skin conditions are described herein and include, but are not limited to: actinic keratosis (AK), malignant freckle-like nevus, leukoplakia of the mucous membranes, and Bowen's disease.

[0509] Exemplary kit In some embodiments, this disclosure relates to packaging or kits containing the microneedles described herein. In some embodiments, this disclosure relates to packaging or kits containing the therapeutic agents described herein. In some embodiments, the kit may also contain additional therapeutic agents for combination therapy with microneedles. In some embodiments, the kit may also contain a disinfectant (e.g., an alcohol swab). In some embodiments, the kit may also contain instructions for use (e.g., instructions for use in applying or administering the microneedle device described herein). In some embodiments, such packaging and kits described herein may be used for vaccination purposes, such as to achieve broad-spectrum immunization in subjects as described herein. In some embodiments, such packaging and kits described herein may be used for cancer treatment or prevention purposes, such as to treat or prevent cancer in subjects as described herein.

[0510] vaccine The microneedles and methods described herein can also be used to deliver vaccines to subjects in need. Regarding vaccine delivery, this disclosure is based, at least in part, on the finding that modulating the kinetics of antigen presentation via controlled and / or sustained-release compositions and devices (e.g., microneedles, such as filament-based microneedles, and microneedle devices) can drive a more potent and / or durable immune response (e.g., a more potent and / or durable cellular and / or humoral immune response) in subjects, compared to single-dose or bolus administration of a vaccine, such as an influenza vaccine as described herein. In some embodiments, controlled or sustained release of a vaccine as described herein can be used to achieve broad-spectrum immunization in subjects.

[0511] In some implementations, the microneedles and microneedle devices described herein exhibit controlled or sustained release of a vaccine (e.g., an influenza vaccine) for at least about 1-2 weeks (e.g., at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days), resulting in one or more of enhanced immunogenicity, enhanced immune response, and / or broad-spectrum immunity.

[0512] In some embodiments, the microneedles of this disclosure can be configured to achieve controlled or sustained release of vaccines, antigens, and / or immunogens (e.g., influenza vaccines) as described herein. Without wishing to be bound by theory, administration of the vaccine-containing microneedles disclosed herein can induce broad-spectrum immunity against viruses in subjects.

[0513] Non-limiting examples of vaccines used in the microneedles and microneedle devices (e.g., microneedle patches) described herein may include commercial vaccines such as seasonal vaccines, pandemic vaccines, and / or universal vaccines; egg-based vaccines; cell culture-based vaccines; recombinant vaccines; live attenuated viruses, inactivated whole viruses, lysed virions, and / or protein subunit vaccines; and adjuvant vaccines.

[0514] As used herein, the term "virus" refers to an infectious agent composed of nucleic acids encased in a protein shell. These infectious agents cannot replicate autonomously (i.e., replication requires the use of the host cell's operational parts). Viral genomics can be single-stranded (ss) or double-stranded (ds) RNA or DNA, and may or may not utilize reverse transcriptase (RT). Additionally, ssRNA viruses can be positive (+) or negative (-). Exemplary viruses include, but are not limited to, dsDNA viruses (e.g., adenoviruses, herpesviruses, poxviruses), ssDNA viruses (e.g., parvoviruses), dsRNA viruses (e.g., Reoviruses), (+)ssRNA viruses (e.g., microRNA viruses, Toga viruses), (-)ssRNA viruses (e.g., orthomyxoviruses, rhabdoviruses), ssRNA-RT viruses (i.e., (+) positive RNA with a DNA intermediate in its life cycle (e.g., retroviruses)), and dsDNA-RT viruses (e.g., hepatotropic DNA viruses). In some implementations, the virus may also include wild-type (natural) viruses, killed viruses, attenuated live viruses, modified viruses, recombinant viruses, or any combination thereof. Exemplary retroviruses include human immunodeficiency virus (HIV). Other examples of viruses include, but are not limited to, enveloped viruses, respiratory syncytial viruses, non-enveloped viruses (e.g., human papillomavirus (HPV)), bacteriophages, recombinant viruses, and viral vectors. As used herein, the term "bacteriophage" refers to a virus that infects bacteria.

[0515] As examples, various commercial influenza vaccines that can be incorporated into the microneedles of this disclosure are listed below. Additionally, influenza vaccines comprising mRNA, DNA, viral vectors, and / or virus-like particles (VLPs) are also suitable for use in the microneedles and microneedle devices (e.g., microneedle patches) described herein. In some embodiments, the influenza vaccine may target matrix protein 1, matrix protein 2 (M2e), and / or nucleoprotein (NP) of the influenza virus.

[0516] Example This disclosure is further described in detail with reference to the following experimental embodiments. These embodiments are provided for illustrative purposes only and are not intended to be limiting, unless otherwise stated. Therefore, this disclosure should not be construed as limited to the following embodiments, but should be construed as covering any and all variations that become apparent as a result of the teachings provided herein.

[0517] Example 1. Efficacy of filament-based microneedle administration of anticancer agents and immunomodulators for the treatment of solid tumors / cancer In vivo evaluation The in vivo model of mouse melanoma can be used to evaluate the efficacy of sustained release of multiple therapeutic agents (e.g., cytokines, checkpoint inhibitors, chemotherapeutic agents, mRNA encoding anticancer agents, or combinations of two, three, or all four) in their ability to control the tumor microenvironment. Although the model used in these experiments was melanoma, the platform is expected to have broader applications in other cancers, including solid tumors.

[0518] Mouse melanoma cells (B16-F10) were injected into the flank (right side) of mice (C57Bl / 6) to induce tumor formation. The drug was then administered intratumorally or peritumorally (i.e., intradermally or subcutaneously): 1. Single push bet, 2. A series of fractionated injections, wherein the total dose administered within the time frame is equal to that of a single bolus injection. These injections may be administered once daily or every other day.

[0519] 3. A microneedle patch formulated with one or more therapeutic agents.

[0520] The therapeutic agents to be studied in these experiments (i.e., as single agents and combinations) may include, but are not limited to: Anticancer agents, such as: Chemotherapy agents: gemcitabine, doxorubicin, oxaliplatin, dacarbazine, temozolomide Targeted therapies: Vemurafenib, Dabrafenib, Trametinib Other: mRNA encoding anticancer agents Immunomodulators, such as: Cytokines: IL-2, IL-12, IL-15, GM-CSF Immunomodulators: CpG, c-di-GMP Checkpoint inhibitors: anti-PD1, anti-PD-L1, anti-CTLA4 Other: mRNAs encoding cytokines and other immune regulatory molecules The duration of sustained release (daily injection or microneedling) will be explored and optimized (~2–28 days). Additionally, multiple cycles of sustained release may be optimal for tumor clearance. To test this, the drug will be administered via a single bolus injection or sustained release (daily injection or microneedling) over approximately ~2–28 days. Animals will be deprived of treatment for a period (~5–14 days), followed by a second round of the drug, identical to the first.

[0521] Tumor growth was measured two to three times per week. The animal was euthanized when humanitarian endpoints were reached. These endpoints included weight loss >15%, physical condition score <1, and tumor volume >1 cm. 3 The humane endpoints are: tumor length (defined as the longest dimension) > 1.5 cm, or tumor ulceration / necrosis. These humane endpoints apply to all in vivo experiments using melanoma models.

[0522] Sustained release of therapeutic agents can lead to inhibition of tumor growth (compared to a conventional single bolus injection) or complete tumor clearance. These experiments will determine the optimal pharmacokinetics, timing of administration, and route of delivery. Importantly, the aim is to narrow down and identify agents or combinations of agents that have an effect on tumor growth. The ability of these agents to enhance ectopic effects and memory responses will then be tested (experiments detailed below).

[0523] Intratumoral administration of IL-2 or gemcitabine in B16-F10 mice Following the methods described above, using a B16-F10 mouse melanoma tumor model with IL-2 or gemcitabine, daily intratumoral administration was compared to a single intratumoral bolus injection. For this study, two cycles of treatment were performed, including bolus injections on days 7 and 21, and daily administration on days 7–11 and 21–25. (See [link to study]) Figure 6A On day 0, mice were inoculated subcutaneously with B16-F10 cells. On days 4 and 9, mice were administered a single dose of anti-PD1 mAb (100 μg per dose) via intraperitoneal injection. Data were collected from both experiments, with 15 mice in each group.

[0524] First cycle On day 7, mice in the 'single bolus' group received a single bolus of IL-2 (5 µg) or gemcitabine (475 µg). Mice in the 'daily administration' group received daily administrations of IL-2 (1 µg) or gemcitabine (95 µg) (intratumorally) from day 7 to day 11 (a total of 5 doses). Each daily administration was approximately one-fifth of the single bolus dose.

[0525] Second cycle On day 21, mice in the 'single bolus injection' group received a single bolus injection of IL-2 (5 µg) or gemcitabine (475 µg). Mice in the 'daily administration' group received daily (intratumoral) administration of IL-2 (1 µg) or gemcitabine (95 µg) from day 21 to 25 (a total of 5 doses). Each daily administration was a fraction of approximately 1 / 5 of the single bolus injection dose.

[0526] Compared with the corresponding mice that received bolus administration, B16-F10 mice that received daily administration of IL-2 had a lower tumor burden, as determined by changes in tumor volume over time (see [link]). Figure 6B (mean + SEM). Mice receiving daily doses of IL-2 also had better survival rates than mice receiving bolus doses of IL-2, as indicated by... Figure 6C This is confirmed by the Kaplan-Meier curve shown in the figure.

[0527] Similarly, B16-F10 mice treated with daily intratumoral administration of gemcitabine showed lower tumor burden compared to their counterparts who received bolus injections of gemcitabine, as determined by changes in tumor volume over time (see [link to relevant documentation]). Figure 6D (mean + SEM). Mice receiving daily intratumoral gemcitabine had higher survival rates than the bolus injection group, as indicated by... Figure 6E The Kaplan-Meier curve provided is shown in the figure.

[0528] Intratumoral administration of gemcitabine in CT26 mice Further studies were conducted in a mouse CT26 colon cancer model using gemcitabine, comparing daily intratumoral administration with a single intratumoral bolus injection (see [link to study]). Figures 7A-7B On day 0, mice were inoculated subcutaneously with CT26 cells. On days 4 and 9, mice were administered a single dose of anti-PD1 mAb (100 µg each) via intraperitoneal injection. Data were collected from 10 mice in each of the two experiments.

[0529] CT26 mice in the 'single bolus injection' group received a single bolus injection of gemcitabine (475 μg) on ​​day 7 (intratumoral) and four doses of saline over the next four days (intratumoral). Figure 7A CT26 mice in the 'daily dosing' group received daily dosing of gemcitabine (95 μg) (intratumoral) from day 7 to day 11 (total of 5 doses). Figure 7B Each daily dose was approximately one-fifth of a single bolus injection. Analysis of tumor volume changes over time revealed that mice in the daily administration group had a lower tumor burden compared to the single bolus injection group (see [link to article]). Figure 7C The overall survival rate of mice in the daily dosing group was also greater than that in the bolus dosing group, such as... Figure 7D This is evidenced by the Kaplan-Meier survival curves provided in the document.

[0530] Example 2. In vivo assessment of ectopic effects in distant tumors To determine whether sustained release produces an ectopic effect—the phenomenon where local treatment of the tumor inhibits further growth of distant tumors or promotes clearance of distant tumors—tumors were induced in two ventral regions (right and left) of mice using B16-F10 melanoma cells. Once tumors had appeared, the agent was administered to the right-sided tumor as follows: 1. Single push bet 2. A series of fractional injections 3. Microneedle patches formulated with pharmaceutical preparations.

[0531] The growth of both tumors was measured over time, two to three times per week. Animals were euthanized when a humane endpoint was reached. If sustained release of the drug promoted ectopic effects, the left-sided (untreated) tumor should be significantly smaller than those in animals treated with a single bolus injection.

[0532] Example 3. In vivo assessment of the ability to establish memory responses that will lead to future tumor rejection To determine whether a superior immune memory response was established after primary tumor treatment, tumors were induced in a single lateral ventral region (right side) of mice using B16-F10 melanoma cells. The following agents were administered: 1. Single push bet 2. A series of fractional injections 3. Microneedle patches formulated with pharmaceutical preparations.

[0533] Tumor size was measured over time. After a prolonged period (~60-90 days), cells were injected into the ventral side (left) opposite the initial tumor. The size of the new tumor was tracked over time. If sustained release of the agent enhanced the anticancer memory response, the new tumor would either fail to grow or be significantly smaller than those in animals that received conventional treatment.

[0534] Example 4. In vivo evaluation of sustained release of cytokines / chemotherapy agents / checkpoint inhibitors For protein-based agents (e.g., cytokines, anti-checkpoint antibodies), the agents are labeled with fluorophores. If a combination of agents is used, each agent is labeled with a different fluorophore. Tumors are induced in mice using the B16-F10 model as previously described. Treatment is administered intratumorally or peritumorally via: 1. Single push bet 2. Microneedle patches formulated with pharmaceutical preparations.

[0535] The duration of drug release is visualized using an in vivo imaging system (IVIS). Drugs administered via bolus injection are rapidly cleared from the tumor, while drugs from microneedles should remain within the tumor for a prolonged period (days to weeks).

[0536] Chemotherapy agents are typically small molecules that cannot be labeled with large fluorophores. To measure the sustained release of these agents using IVIS, alternative molecules that are similar to one or more agents (e.g., rhodamine B, coumarin 6) in terms of solubility, size, and charge are administered to the tumor, as previously described.

[0537] Example 5. In vitro evaluation of the efficacy of filament-based microneedle delivery of cytokines in the treatment of melanoma. To determine the release and biological function of cytokine "cargo" from filament-based microneedles, a series of in vitro assays were used.

[0538] For all cytokines (e.g., IL-2, IL-12, IL-15, GM-CSF), the amount of cytokines released from the microneedles was determined using a commercially available enzyme-linked immunosorbent assay (ELISA).

[0539] To assess whether biological function is preserved, cell line-based assays are used. For selected cytokines, cell lines whose growth depends on these cytokines are obtained (e.g., CTLL-2, HT-2). Cell proliferation is measured over several days (2–5 days) using a tetrazolium salt (e.g., WST-1, MTT, or MTS).

[0540] IL-12 function can be measured by IFNα produced by mouse spleen cells. IFNα was then measured using ELISA.

[0541] The biological functions of most cytokines can be determined using commercially available reporter cell lines that are genetically engineered to produce measurable proteins in a dose-dep...

Claims

1. A microneedle device (e.g., a microneedle patch) comprising a plurality of silk fibroin-based microneedles, wherein the plurality of microneedles comprises: The first microneedle containing anticancer agents; and The second microneedle contains an immunomodulator. The first and / or second microneedles contain silk fibroin (e.g., regenerated silk fibroin and / or recombinant silk fibroin), and the microneedle device is configured to deliver the anticancer agent and the immunomodulator to the subject.

2. The microneedle device according to claim 1, wherein the first and / or second microneedle among the plurality of microneedles comprises: (i) Substrate (e.g., a soluble substrate), (ii) A silk fibroin tip (e.g., an implantable silk fibroin tip), said silk fibroin tip comprising silk fibroin applied to the substrate, and (iii) (Optional) Applying a backing to the substrate.

3. Multiple microneedles, wherein the multiple microneedles comprise: The first microneedle containing anticancer agents; and The second microneedle contains an immunomodulator. The first and / or second microneedles contain silk fibroin, such as regenerated silk fibroin and / or recombinant silk fibroin.

4. The microneedle device according to claim 2, wherein the silk fibroin tip comprises the anticancer agent and / or the immunomodulator.

5. The microneedle device of claim 2, wherein the substrate comprises the anticancer agent and / or the immunomodulator.

6. The microneedle device or multiple microneedles according to any one of the preceding claims, wherein the microneedles are configured to pierce a biological barrier (e.g., skin).

7. The microneedle device or plurality of microneedles according to any one of the preceding claims, wherein the microneedle device or plurality of microneedles further comprises a third microneedle, optionally wherein the third microneedle comprises an anticancer agent and / or an immunomodulator.

8. The microneedle device or plurality of microneedles according to any one of the preceding claims, the microneedle device or plurality of microneedles being configured to deliver (e.g., release) two or more anticancer agents (e.g., three or more, four or more, or five or more anticancer agents).

9. The microneedle device or multiple microneedles according to claim 8, wherein the two or more anticancer agents are in the same microneedle.

10. The microneedle device or multiple microneedles according to claim 8, wherein the two or more anticancer agents are in different microneedles.

11. The microneedle device or plurality of microneedles according to any one of the preceding claims, said microneedle device or plurality of microneedles being configured to deliver (e.g., release) two or more immunomodulators (e.g., three or more, four or more, or five or more immunomodulators).

12. The microneedle device or multiple microneedles according to claim 11, wherein the two or more immunomodulators are in the same microneedle.

13. The microneedle device or multiple microneedles according to claim 11, wherein the two or more immunomodulators are in different microneedles.

14. The microneedle device or multiple microneedles according to any one of the preceding claims, wherein the anticancer agent and the immunomodulator are in the same microneedle.

15. The microneedle device or multiple microneedles according to any one of the preceding claims, wherein the anticancer agent and the immunomodulator are in different microneedles.

16. The microneedle device or multiple microneedles according to any one of the preceding claims, wherein the anticancer agent is selected from one or more of small molecules (e.g., chemotherapeutic drugs), biological agents (e.g., antibodies), viral cancer therapeutics, nanomedicines, and nucleic acid molecules (e.g., DNA and / or RNA).

17. A microneedle device or multiple microneedles according to any one of the preceding claims, wherein the anticancer agent is mRNA, optionally wherein the mRNA encodes an anticancer agent and / or an immunomodulator, optionally wherein the mRNA encodes a checkpoint inhibitor, a TLR agonist, a STING agonist, a RIG agonist, a cancer vaccine, a targeted therapy, and / or a cytokine.

18. The microneedle device or multiple microneedles according to any one of the preceding claims, wherein the immunomodulator is selected from checkpoint inhibitors, Toll-like receptor (TLR) agonists, STING agonists, RIG agonists, cancer vaccines, and cytokines.

19. The microneedle device or multiple microneedles according to claim 18, wherein the checkpoint inhibitor inhibits a checkpoint molecule selected from the group consisting of: CTLA4, PD1, PD-L1, PD-L2, TIM3, LAG3, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), BTLA, KIR, MHC class I, MHC class II, GAL9, VISTA, BTLA, TIGIT, LAIR1, and A2aR.

20. The microneedle device or multiple microneedles according to claim 17 or 18, wherein the TLR agonist is selected from TLR-1 agonist, TLR-2 agonist, TLR-3 agonist, TLR-4 agonist, TLR-5 agonist, TLR-6 agonist, TLR-7 agonist, TLR-8 agonist, TLR-9 agonist, TLR-10 agonist, TLR-1 / 2 agonist, TLR-2 / 6 agonist, or TLR-7 / 8 agonist.

21. The microneedle device or multiple microneedles according to claim 17 or 18, wherein the STING agonist is a cyclic dinucleotide, such as a cyclic dinucleotide containing a purine or pyrimidine nucleobase (e.g., adenosine, guanine, uracil, thymine, or cytosine nucleobase), optionally bis-(3'-5')-cyclic diguanosine monophosphate (c-di-GMP).

22. The microneedle device or multiple microneedles according to claim 17 or 18, wherein the cytokine is GM-CSF, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-15, IL-18, IL-21, IFN-α, IFN-β, IFN-γ, MIP-1α, MIP-1β, TGF-β, TNF-α, or TNFβ.

23. The microneedle device or plurality of microneedles according to any one of the preceding claims, said microneedle device or plurality of microneedles being configured to administer anti-PD1 antibody and / or anti-CTLA4 antibody in combination with one or more of the following: (i) IL-2; (ii) IL-12; (iii) IL-15; (iv) IL-18; (v) Gemcitabine (GEMZAR®); (vi) Vemurafenib (ZELBORAF®); (vii) Dabrafenib (TAFINLAR®); (viii) Trametinib (MEKINIST®); (ix) Dorothycin®; (x) c-di-GMP; (xi) mRNA; (xii) TLR-9 agonists (e.g., unmethylated CG dinucleotides (CpG ODN)); (xiii) Oxaliplatin; and (xiv) GM-CSF.

24. A microneedle device or a plurality of microneedles according to any one of the preceding claims, wherein the device or the plurality of microneedles is configured for sustained release of the anticancer agent and / or the immunomodulatory agent, and wherein the sustained release is over a period of time including at least about 2 days (e.g., about 2, 3, 4, 5, 6, 7 or more days, e.g., about 5 to about 10 days, e.g., about 7 to about 15 days, e.g., about 1 week to about 2 weeks, about 1 week to about 3 weeks, or about 2 weeks to about 4 weeks, e.g., about 1 month to about 3 months, e.g., about 2 months to about 4 months, e.g., about 3 months to about 6 months).

25. A microneedle device or a plurality of microneedles according to any one of the preceding claims, wherein the device or the plurality of microneedles is configured for abrupt release of the anticancer agent and / or the immunomodulatory agent, and wherein the abrupt release is within a time period including at least about 1 hour (e.g., about 1 to about 30 minutes, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or 24 hours).

26. The microneedle device or plurality of microneedles according to any one of claims 8-15, 24 or 25, wherein the release of the anticancer agent occurs at a different rate than the release of the immunomodulator, such that the anticancer agent is released substantially before or substantially after the release of the immunomodulator.

27. The microneedle device or plurality of microneedles according to any one of the preceding claims, the microneedle device or plurality of microneedles being configured to be applied (e.g., administered) to a biological barrier selected from the skin layer, cell membrane, mucous surface, oral cavity, or buccal cavity.

28. The microneedle device or plurality of microneedles according to any one of the preceding claims, the microneedle device or plurality of microneedles being configured to be applied (e.g., administered) to a tumor (e.g., a metastatic tumor).

29. The microneedle device or plurality of microneedles according to any one of the preceding claims, the microneedle device or plurality of microneedles being configured to be applied (e.g., administered) to a tumor site after tumor resection, for example to induce an immune response to the tumor and / or ablate any cancer cells remaining after resection.

30. The microneedle device or plurality of microneedles according to any one of the preceding claims, the microneedle device or plurality of microneedles being configured to be applied (e.g., administered) to a tumor site prior to tumor resection, for example to induce an immune response to the tumor and / or to ablate any cancer cells remaining after resection.

31. The microneedle device or plurality of microneedles according to any one of the preceding claims, the microneedle device or plurality of microneedles being configured to be applied (e.g., administered) within a tumor.

32. The microneedle device or plurality of microneedles according to any one of the preceding claims, the microneedle device or plurality of microneedles being configured to apply (e.g., administer) around a tumor.

33. The microneedle device or plurality of microneedles according to any one of the preceding claims, the microneedle device or plurality of microneedles being configured to be applied (e.g., administered) to a skin lesion or adjacent skin lesion (e.g., skin lesion associated with cancer or precancerous conditions).

34. The microneedle device or plurality of microneedles according to any one of the preceding claims, wherein the local and / or systemic delivery (e.g., release) results in: (i) Inhibition of tumor growth at or near the application site; (ii) Induction of a local immune response to ablate tumors at or near the application site; (iii) An increase in activated immune effector cells (e.g., T cells) in the tumor microenvironment; (iv) Decrease in local immunosuppressive cells (e.g., regulatory T cells (Treg)); (v) Induction of a systemic immune response to ablate tumors at distant sites; (vi) Immune memory of cancer or precancerous conditions; and / or (vii) Immune response to tumor antigens such as neoantigens; and / or (viii) Prevention and / or suppression of cancer recurrence (e.g., cancer relapse).

35. The microneedle device or multiple microneedles of claim 2, wherein the backing is selected from solid supports, such as paper-based materials, plastic materials, polymer materials, or polyester-based materials (e.g., Whatman 903 paper, polymer tape, plastic tape, adhesive-backed polyester tape, or other medical tape).

36. The microneedle device or plurality of microneedles according to any one of claims 2, 5, or 35, wherein the substrate (e.g., a soluble substrate) comprises two or more of the following: (i) Polysaccharides (e.g., dextran); (ii) Disaccharides (e.g., sucrose, maltose, and trehalose); (iii) Polymers (e.g., methylcellulose, polyethylene glycol (PEG), carboxymethyl cellulose (CMC), polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), and hyaluronic acid salts); (iv) Proteins (e.g., gelatin); (v) Plasticizers (e.g., glycerin, propylene glycol); and (vi) Surfactants (e.g., octylphenol ethoxylates (e.g., Triton-X), polysorbates, poloxamer and / or polyethoxylated alcohols).

37. The microneedle device or multiple microneedles according to any one of claims 2, 5, 35 or 36, wherein the substrate comprises one or more of the following: gelatin, dextran, glycerol, polyethylene glycol (PEG) (e.g., including low molecular weight PEG), sucrose, trehalose, maltose, carboxymethyl cellulose (CMC), polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), hyaluronic acid salt, methylcellulose and / or surfactants (e.g., octylphenol ethoxylate (e.g., Triton-X), polysorbate, poloxamer such as P188, and / or polyethoxylated alcohols), optionally wherein the microneedles are configured for sustained release and / or sudden release.

38. The microneedle device or plurality of microneedles according to claim 2 or 4, wherein the silk fibroin tip comprises two or more of the following: (i) Disaccharides (e.g., sucrose, maltose and trehalose); (ii) Polymers (e.g., methylcellulose, polyethylene glycol (PEG), carboxymethyl cellulose (CMC), polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), hyaluronic acid salts); (iii) Amino acids (e.g., threonine); (iv) Plasticizers (e.g., glycerin, propylene glycol); and (v) Buffers (e.g., PBS).

39. The microneedle device or multiple microneedles according to any one of claims 2, 4 or 38, wherein the silk fibroin tip comprises an excipient.

40. The microneedle device or multiple microneedles according to any one of claims 2, 4, 38 or 39, wherein the silk fibroin tip comprises one or more of carboxymethyl cellulose (CMC), sucrose and threonine.

41. The microneedle device or multiple microneedles according to any one of claims 2, 4 or 38-40, wherein the silk fibroin tip comprises a 60 MB silk fibroin solution of about 1% w / v to about 10% w / v (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10% w / v), or a silk fibroin solution according to Figure 5, for example, a 100 kDa to 200 kDa (e.g., about 153 kDa) silk fibroin solution.

42. The microneedle device or multiple microneedles according to any one of claims 2, 4 or 38-41, wherein the silk fibroin tip comprises a 180 MB silk fibroin solution of about 1% w / v to about 10% w / v (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10% w / v), or a silk fibroin solution according to Figure 5, for example, a 36 kDa to 100 kDa (e.g., about 71 kDa) silk fibroin solution.

43. The microneedle device or plurality of microneedles according to any one of claims 2, 4, or 38-42, wherein the microneedle device or plurality of microneedles contains silk fibroin in an amount of about 0.5 µg to about 500 µg, optionally, about 0.5 µg to about 5 µg, or about 1 µg to about 10 µg, or about 5 µg to about 15 µg, or about 10 µg to about 20 µg, or about 15 µg to about 25 µg, or about 20 µg to about 30 µg, or about 25 µg to about 35 µg, or about 30 µg to about 40 µg, or about 35 µg to about 45 µg, or about 40 µg to about 50 µg, or about 45 µg to about 55 µg, or about 50 µg to about 60 µg, or about 55 µg... g to about 65µg, or about 60µg to about 70µg, or about 65µg to about 75µg, or about 70µg to about 80µg, or about 75µg to about 85µg, or about 80µg to about 90µg, or about 85µg to about 95µg, or about 90µg to about 100µg, or about 95µg to about 150µg, or about 125µg to about 175µg, or about 150µg to about 200µg, or about 225µg to about 275µg, or about 250µg to about 300µg, or about 325µg to about 375µg, or about 350µg to about 400µg, or about 425µg to about 475µg, or about 450µg to about 500µg silk fibroin.

44. The microneedle device or plurality of microneedles according to any one of claims 2, 4 or 38-43, wherein the microneedle device or plurality of microneedles contains silk fibroin in an amount of about 1% to about 75% by weight, optionally about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75% by weight of silk fibroin.

45. The microneedle device or multiple microneedles according to any one of claims 2, 4 or 38-44, wherein the microneedles are configured to implant the silk fibroin tip into the subject's biological barrier at a depth of about 100 μm to about 1 mm (e.g., the maximum penetration depth of the distal portion of the tip).

46. ​​A method of treating cancer (e.g., metastatic cancer) and / or inducing an immune response to cancer (e.g., metastatic cancer), said method comprising contacting (e.g., administering) a microneedle device or plurality of microneedles according to any one of the preceding claims with (e.g., administering) a cancer (e.g., metastatic tumor) site of a subject, thereby producing one or more of the following: (i) Lysing cancer cells, such as tumor cells, to release cancer-associated antigens (e.g., neoantigens) and / or exposing cancer-associated antigens (e.g., neoantigens) to the subject's immune system; (ii) Cancer-associated antigens (e.g., neoantigens) that are complexed with the major histocompatibility complex (MHC) are presented on the surface of immune system cells (e.g., helper cells such as B cells, dendritic cells, etc.) by antigen-presenting cells (APCs). (iii) Cancer-associated antigens (e.g., neoantigens) that are recognized by immune effector cells (e.g., T cells and / or NK cells); (iv) Activating and / or expanding immune effector cells, such as T cells and / or NK cells, that are specific for cancer-associated antigens (e.g., neoantigens) displayed in the subject; and (v) Enhance (e.g., stimulate or upregulate) the immune response of immune effector cells such as T cells and / or NK cells, which promote the killing of target cells expressing the cancer-associated antigen (e.g., neoantigen) in the subject and / or inhibit the growth or proliferation of the target cells.

47. A method for treating cancer (e.g., metastatic cancer) and / or inducing an immune response to cancer (e.g., metastatic cancer), the method comprising bringing a microneedle device or a plurality of microneedles according to any one of claims 1-45 into contact (e.g., application) a site of cancer (e.g., metastatic tumor) and / or skin condition (e.g., dermatitis) in a subject.

48. A method of treating cancer (e.g., metastatic cancer) or precancerous conditions (e.g., precancerous skin conditions), the method comprising bringing a microneedle device or a plurality of microneedles according to any one of claims 1-45 into contact (e.g., application) a tumor (e.g., metastatic tumor) or lesion (e.g., skin lesion) of a subject.

49. A method of treating cancer (e.g., metastatic cancer), the method comprising bringing a microneedle device or plurality of microneedles according to any one of claims 1-38 into contact (e.g., application) with a tumor (e.g., metastatic tumor) site in a subject, thereby inducing: (i) Local immune response and / or local cytotoxicity to the cancer (e.g., evidenced by death of cancer cells at or near the site of microneedling application, such as reduction in local tumor size and / or local tumor burden); and / or (ii) Distal immune response and / or distal cytotoxicity to the cancer (e.g., as demonstrated by the death of cancer cells at a location distal to where the microneedle patch was applied, such as a reduction in distal tumor size and / or overall tumor burden).

50. The method according to any one of claims 46-49, wherein the cancer is selected from: anal cancer; basal cell carcinoma; bladder cancer; bone cancer; brain tumor; breast cancer; cervical cancer; colon and rectal cancer; endometrial cancer; esophageal cancer; gastrointestinal stromal tumor; gestational trophoblastic disease; head and neck cancer; Hodgkin lymphoma; Kaposi's sarcoma; renal (renal cell) carcinoma; leukemia; liver cancer; lung cancer; malignant mesothelioma; melanoma; Merkel cell carcinoma; multicentric Castleman's disease; multiple myeloma and other plasma cell tumors; myeloproliferative neoplasms; neuroblastoma; non-Hodgkin lymphoma; Ovarian cancer, fallopian tube cancer, or primary peritoneal cancer; Pancreatic cancer; penile cancer; pheochromocytoma and paraganglioma; prostate cancer; retinoblastoma; rhabdomyosarcoma; skin cancer; squamous cell carcinoma; soft tissue sarcoma; solid tumors anywhere in the body; stomach cancer; testicular cancer; thyroid cancer; vaginal cancer; vulvar cancer; And Wilms' tumor and other childhood kidney cancers.

51. The method according to any one of claims 46-50, wherein the cancer is melanoma.

52. The method according to any one of claims 46-50, wherein the cancer is basal cell carcinoma.

53. The method according to any one of claims 46-50, wherein the cancer is squamous cell carcinoma.

54. The method according to any one of claims 46-50, wherein the cancer is Merkel cell carcinoma.

55. The method according to any one of claims 46-50, wherein the cancer is breast cancer.

56. The method of claim 48, wherein the precancerous condition is a precancerous skin condition, optionally selected from actinic keratosis (AK), malignant freckle-like nevus, leukoplakia of the mucosa, and Bowen's disease.

57. The method according to any one of claims 46-56, wherein the contact (e.g., application) is performed within a tumor.

58. The method according to any one of claims 46-56, wherein the contact (e.g., application) is performed around the tumor.

59. The method according to any one of claims 46-56, wherein the contact (e.g., application) is performed prior to surgical resection.

60. The method according to any one of claims 46-56, wherein the contact (e.g., application) is performed after surgical excision.

61. The method according to any one of claims 46-60, wherein the contact (e.g., application) is performed in combination with standard care treatment (e.g., standard care treatment for cancer or precancerous conditions), the standard care treatment optionally selected from surgery, chemotherapy, immunotherapy, targeted therapy, hormone therapy and / or radiation therapy.

62. The method of claim 61, wherein the standard care treatment is performed before, after, or simultaneously with the microneedle device.

63. The method according to any one of claims 46-62, wherein the subject is a human subject.

64. A method for manufacturing a microneedle device, the method comprising: A mold is provided comprising a mold body having an array of needle cavities of predetermined shapes formed therein, such as pyramidal and / or conical needle cavities; The tip of the needle cavity is filled with a composition comprising a solution of silk fibroin and a therapeutic agent (e.g., an anticancer agent, an immunomodulator, or both); The filling tip of the needle cavity is dried to produce a silk fibroin tip, and the silk fibroin tip is optionally annealed. The needle cavity of the mold is further filled with a first base (e.g., a soluble base) solution; The first substrate solution is dried to form a first substrate layer; (Optionally) One or more additional base layers are formed by adding one or more additional base solutions to the first base layer and drying the additional base solutions, wherein the additional base solutions are different from the first base solution. (Optionally) A backing is applied to the substrate layer (e.g., the first substrate layer, or one or more other substrate layers) to fabricate a microneedle device.

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