Applicator for medicine patch

Abstract

A patch applicator device is provided. The device may include a reusable or disposable system for applying a push-out or split patch to the skin of a patient. The device can improve application and effectiveness of a microneedle-based patch.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This invention was made with government support under R44AI142948 and SB1AI164584 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] This application claims priority under 35 USC §119 to U.S. Provisional Application No. 63 / 355,301, filed on June 24, 2022. The entire contents of the provisional application are incorporated herein by reference. Technical Field

[0004] The present disclosure generally relates to an applicator device and system for a drug patch. The device and system can be used to securely apply the patch to a patient's skin. Background Art

[0005] There are many devices currently available or in development for delivering medications (e.g., flu vaccines and / or coronavirus vaccines, e.g., mRNA-based vaccines) to patients through the skin. The simplest of these devices can include systems that release or apply substances to the skin for absorption. More complex systems can facilitate delivery through various techniques, such as the use of small needles to facilitate delivery deeper within the skin, or other systems such as iontophoresis to facilitate movement of materials into the skin.

[0006] Recently, patch-type devices have been developed that place biodegradable needle-like devices into the skin. These devices can carry the substance to be delivered and slowly degrade over time, exposing the body to the delivered substance for the desired period of time, and possibly other agents that protect the substance to be delivered or provide other therapeutic benefits (e.g., control release rate, improve biological function). Such devices can be used to deliver vaccines, small molecule drugs, biologics, combination products, or other therapeutic or preventive substances.

[0007] The effectiveness of such biodegradable needle patches can be improved by ensuring that the patch is securely applied with sufficient force to place the biodegradable needles to the desired depth within the skin. However, while such patches can be effectively applied by a patient or health care provider through simple manual application, it would be beneficial to provide an improved system for securely applying biodegradable patches, requiring little or minimal training, and with a high level of repeatability.

[0008] Thus, the present disclosure provides improved devices for applying medical patches, including biodegradable needle patches. Summary of the invention

[0009] The present invention relates to an applicator device for applying a patch-type device to a patient's skin. The device can allow for secure application of the patch, including uniform and secure application, thereby obtaining sufficient force and / or skin penetration depth to ensure that the needle-like portion of the patch is located at a desired depth within or under a portion of the skin. The applicator can be configured to provide a predetermined force to quickly and securely apply the patch to a desired location, thereby facilitating the use of a selected patch to improve the delivery of an active agent (e.g., a drug, vaccine, biologic, or other material).

[0010] The applicator may include an outer body portion having a substantially cylindrical shape and a hollow interior; a piston slidably connected to the hollow interior of the outer body portion; a compressible member located within the outer body and configured to apply downward pressure to the piston; and an actuator located below the piston and slidably engaged with the hollow interior and extending from the bottom of the outer body so that upward pressure on the actuator pushes the piston upward and compresses the compressible member. The piston and the hollow interior of the outer body portion are slidably connected by a protrusion and a cam path, and the cam path forms a continuous loop so that upward pressure on the piston causes the piston to compress the compressible member, moving the piston upward into the hollow interior of the outer body portion until the protrusion reaches the top of the cam path and engages the downward portion of the cam path, thereby releasing the piston to the downward portion of the continuous loop to release the piston and push the piston downward. The drug patch is held in an annular holder at the bottom of the actuator and is pushed downward through the annular holder and onto an object that is in contact with the bottom surface of the annular holder. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the present invention and, together with the description, serve to explain the principles of the present invention.

[0012] Figure 1 A perspective view of a drug patch applied to a patient's arm.

[0013] Figure 2A and Figure 2B Respectively Figure 1 Side and bottom perspective views of an exemplary applicator system for use with a drug patch are shown.

[0014] Figure 3 is a perspective view of an exemplary applicator showing internal components.

[0015] Figure 4A for Figure 3 Side view of the illustrated embodiment with the compressible member and piston in a downward position.

[0016] Figure 4B and Figure 4C for Figure 3 and Figure 4A Additional views of an exemplary applicator of in a partially compressed position prior to deployment.

[0017] Figure 4D yes Figure 3 , Figure 4A , Figure 4B and Figure 4C Additional views of the exemplary applicator are shown after releasing the piston to deploy the device and apply the patch.

[0018] FIG. 5A to FIG. 5C 1 and 2 are perspective, top and bottom views of a ring-shaped holder for a drug patch according to an exemplary embodiment.

[0019] Fig. 6A and Figure 6B are exemplary patch configurations for use with the disclosed applicators and patch systems.

[0020] Figure 7 A side view of one embodiment of a drug patch ring holder connected to a patch applicator actuator.

[0021] Figure 8 A perspective view of a drug patch retaining mechanism for use with a patch applicator.

[0022] Fig. 9A A perspective view of another retention mechanism for a drug patch for use with a patch applicator.

[0023] Fig. 9B and Fig. 9C for Fig. 9A Side cutaway and exploded views of the retention mechanism shown.

[0024] Fig. 10A and Fig. 10B is a side view showing the movement path and connection mechanism of the piston of an exemplary patch applicator device.

[0025] Fig.11A and Fig. 11B 2 are perspective and side views illustrating the connection and movement of an actuator within an exemplary patch applicator device.

[0026] Fig.12 for Fig.11A and Fig. 11B A perspective view of the actuator shown

[0027] Fig.13A is a perspective view of a piston of an exemplary patch applicator device.

[0028] Fig. 13B is a top view of a piston of an exemplary patch applicator device.

[0029] FIG. 14A to FIG. 14C The interaction between the actuator, outer body portion and piston of an applicator is shown according to various embodiments.

[0030] Fig.15 An alternative release mechanism for securing a drug patch in a patch applicator.

[0031] FIG. 16A to FIG. 16C is an alternative exemplary embodiment of a drug patch backing portion. DETAILED DESCRIPTION

[0032] Reference will now be made in detail to certain exemplary embodiments according to the present disclosure, some examples of which are illustrated in the accompanying drawings. Throughout the drawings, the same reference numerals are used wherever possible to refer to the same or like parts.

[0033] In this application, the use of the singular includes the plural, unless otherwise stated. In this application, the use of "or" means "and / or" unless otherwise stated. In addition, the use of the term "includes" and other forms such as "includes" and "included" is not limiting. Any range described herein will be understood to include the endpoints and all values ​​between the endpoints.

[0034] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including but not limited to patents, patent applications, articles, books, and treatises, are hereby expressly incorporated by reference in their entirety for any purpose.

[0035] Although the principles of the present disclosure are described herein with reference to the illustrative embodiments of specific applications, it should be understood that the present invention is not limited thereto. Those of ordinary skill in the art and those who understand the teachings provided herein will recognize that additional modifications, applications, embodiments, and replacements of equivalents all fall within the scope of the embodiments described herein. Therefore, the present invention should not be considered to be limited by the foregoing description.

[0036] As mentioned above, various patch devices can be used to deliver drugs or other substances into or through the skin. As used herein, "drug" or "drug patch" will be understood to refer to any substance or a patch carrying a substance to provide a biological effect to the patient. "Drug" will be understood to refer to any drug, small molecule drug, vaccine (including any vaccine, such as mRNA vaccine, protein, glycoprotein, live virus, attenuated live virus, inactivated virus, recombinant or other vaccine), peptide, biological product, antibody, vitamin, mineral, hormone or other material that can be delivered into or through the skin.

[0037] The drug patch may include a microneedle-based device, which will be described in detail below. Such a microneedle device may include one or more (preferably a group or an array) microneedles. The microneedles are located on the skin-facing surface of a flexible or semi-rigid patch, and by applying the patch to the patient's skin, the needles can penetrate the skin to a desired depth. In some cases, the microneedles may include a biodegradable component that is deposited at a desired distance in the skin and can be degraded at a desired rate to release or present the drug to the patient, thereby inducing a desired response, such as an immune response to a vaccine. More details about exemplary patches, including microneedle patches, will be further described below.

[0038] To improve the efficacy of any drug patch, it is desirable to ensure proper application of the patch, including uniform application to ensure that as many microneedles as possible are at the appropriate depth. In addition, it is desirable to ensure that the patch is applied so that the microneedles are pushed into the skin to the desired depth without inadvertently shearing off the microneedles above the skin. Proper application of the patch can improve overall efficacy.

[0039] In order to improve the consistency and effectiveness of patch application, an automatic applicator device may be required. Therefore, the present disclosure provides an embodiment of an applicator device having one or more features or advantages over existing devices or simple manual application. The disclosed device can be configured for repeated use or single use, and the device can be pre-loaded with a patch (i.e., as a kit or patch product including an applicator and a patch). Alternatively, the applicator can be separate from the patch, and the patch can be selected and loaded onto the device (e.g., based on the desired medication, patient characteristics, or the need for additional application for more than one patient or for a patient who needs more than one patch).

[0040] The applicator can improve the application of the patch by one or more of the following ways: (1) properly holding and / or stretching / pre-stretching the skin to receive the patch, (2) applying a reliable degree and / or depth of force to the skin to ensure correct microneedle placement, and / or (3) controlling the distribution of the application force on the patch. The structure and function of the applicator in various embodiments will be described in more detail below.

[0041] Figure 1 1 is a perspective view of a medicament patch 10 applied to a patient's arm. As shown, the patch 10 is substantially square, or a square with rounded corners, but other shapes and configurations are also conceivable and described below. The patch usually has an adhesive to fix the patch to the patient's skin. At least the backing layer will be semi-flexible and can be deployed using an applicator as described herein.

[0042] Figure 2A and Figure 2B Respectively Figure 1The side view and the perspective view of the exemplary applicator 100 of shown medicine patch 10 supporting use.As shown in the figure, applicator 100 has upper body part 110 and lower patient contact part 105 (for example, the bottom surface 120 of actuator 122 or annular retainer 50, as described below).Upper body part 110 can be held by the user, and patient contact part 105 can be pressed against the patient's skin.When this device is pressed on the patient's skin, potential energy is generated, until enough pressures are applied, and at this moment, the internal piston system is pushed down to the top of patch 10, so that patch is correctly launched on the patient's skin.The specific details of the applicator internal parts and its function will be described in the following accompanying drawings.

[0043] Figure 3 and FIG. 4A to FIG. 4D The detailed assembly components and operation of the applicator 100 according to various embodiments are illustrated. Specifically, Figure 3 is a perspective view of an exemplary applicator 100 showing internal components. Figure 3 There is shown a patch applicator 100 comprising a patch 10 in the form of a generally flexible sheet.

[0044] The applicator 100 includes an outer body portion 110 that is generally cylindrical and has a hollow interior 112. A piston 114 is slidably coupled to the hollow interior 112 of the outer body portion. A compressible member 118 ( Fig. 10A 1 ) is located within the outer body 110 and is configured to apply downward pressure to the piston 114. An actuator 122 is located below the piston 114 and is slidably engaged with the hollow interior 112. The actuator extends from the outer body 100 so that upward pressure on the bottom region 120 of the actuator 122 pushes the piston 114 upward and compresses the compressible member 118.

[0045] The compressible member 118 can be any suitable spring or other compressible structure. For example, the spring can be a typical spring, a compression spring, a wave spring, a dome spring or a leaf spring. Alternatively, a compressible member such as a balloon, a compressible air bag or a similar structure can be used.

[0046] As reference FIG. 4A to FIG. 4DAs described, the piston 114 and the hollow interior 112 of the outer body portion are slidably connected with the protrusion 130 and the cam path 140, and the cam path forms a continuous ring, so that the upward pressure on the piston causes the piston 114 to compress the compressible member 118, moving the piston upward into the hollow interior of the outer body portion until the protrusion reaches the top of the cam path and engages the downward portion of the cam path, thereby releasing the piston to the downward portion of the continuous ring to release the piston and force the piston downward. In addition, the drug patch 10 is held in an annular holder near the bottom of the actuator 122, and when the applicator is actuated, the drug patch 10 is pushed downward through the annular holder and reaches an object that is in contact with the bottom surface of the annular holder.

[0047] Now go to FIG. 4A to FIG. 4D , to explain the operation of the applicator components, Figure 4A for Figure 3 Side view of the illustrated embodiment with the compressible member 118 and piston 114 in a down position. Figure 4B and Figure 4C is in a partially compressed position before expansion Figure 4A Additional views of an exemplary applicator. Figure 4D yes Figure 3 , Figure 4A , Figure 4B and Figure 4C Additional views of an exemplary applicator after releasing the piston to deploy the device and apply the patch.

[0048] As shown, the piston 114 has a protrusion 130 on at least one side that engages with a cam path 140 on the inner surface of the outer body 110. The protrusion 130 is also Fig.13A , which provides additional individual details of the piston embodiment. As shown, the protrusion 130 is located near the top 132 of the piston 114, but the location of the protrusion can be changed. In some cases, and typically, the piston 114 will include more than one protrusion, and each protrusion will engage a separate cam path 140 on the inner surface of the outer body 110. For example, in one embodiment, the piston will have two protrusions, such as Fig.13A As shown, the two protrusions will be located on opposite sides of the piston. In this way, each protrusion will engage a separate cam path 140 on opposite sides of the inner surface of the outer body 110. It is contemplated that more than two protrusions and cam paths may be used (e.g., three or more protrusion spaces evenly distributed around the piston, with an equal number of cam paths). However, two protrusions will typically be used, as such an embodiment will adequately control the movement of the piston 114.

[0049] Now turn to the specific configuration of the cam path 140. The movement of the protrusion 114 in the cam path 140 is FIG. 4A to FIG. 4DAs shown, the cam path forms a continuous loop. For example, as shown, the loop includes a first upward portion 141, a second upwardly inclined portion 142, a third downwardly inclined portion 143, and a fourth downwardly inclined portion 144. Segments 141 to 144 are ordered based on the sequence of movement during normal operation of the applicator 100. The motion path of the protrusion 130 and the cam path 140 is Fig. 10A and Fig. 10B Shown in a larger view.

[0050] Figure 4A The applicator 100 is shown before use with the actuator 122 in the downward position. As shown, the protrusion 130 is located at the lowest point of the cam path 140, and therefore, the piston 122 is in the low position. When upward pressure is applied to the actuator 122, that is, by pushing the device against the patient's skin, the actuator moves upward and reaches the extension 126 ( Fig.12 ) engages the flange or widened area 138 of the piston 114, such as Fig.13A As pressure on the actuator 122 continues, the piston 114 begins to move upward along the first portion of the cam path 141, thereby compressing the compressible member 118. As the upward pressure continues, the protrusion 130 of the piston moves along the second portion 142 of the cam path 140, and thus, the piston 114 continues to move further upward to compress the compressible member.

[0051] Finally, if Figure 4C As shown, the protrusion is close to the third downward portion 143 of the cam path. When the third downward portion 143 of the cam path is reached, the protrusion 130 is able to move downward because the cam path is downward, and when the protrusion 140 reaches the third downward portion 143 of the cam path 140, the actuator extension 126 is disengaged from the flange or widened area 138 of the piston. The specific structure and function of the piston 114 and the actuator 122 will be explained in more detail below. In some cases, the device 100 can be reset or configured for additional use. In some cases, the retainer is removed and the actuator is pulled to reposition and reset the device. In some cases, the device is configured to allow only a single use.

[0052] like Figure 4D As shown, when the piston moves downward, the piston 114 engages with the patch 10, quickly applying enough pressure to the patch to push the patch 10 through the annular retainer of the applicator to the patient's skin. The actual force applied can vary according to the specific patch structure and the target area of ​​the patient. Typically, the piston will apply a force of about 90N to 130N to the patch against the target area of ​​the patient's skin.

[0053] Although the cam path 140 is illustrated as four sections forming a loop shaped like a parallelogram, it is contemplated that the cam path 140 may have other shapes. For example, any shape that forms a loop to allow for repeated movement of the protrusion through the cam path 140 (i.e., to allow for repeated use of the applicator 100) may be used. For example, a suitable shape may be more circular and closer to an ellipse, but generally the cam path should have a downward portion similar to the fourth section 143, because it is the rapid movement of the protrusion along the fourth section 143 that allows the piston 114 to move rapidly downward to push the patch downward out of the applicator.

[0054] Typically, to correctly position the patch relative to the target area of ​​the patient's skin, and provide a configuration in which a piston 114 can push the patch away from the applicator 100, and the patch 10 is maintained in an annular retainer. Specifically, the patch is maintained by a retainer having a substantially open area and a rigid edge or periphery. The patch is fixed to the inner edge of the annular retainer at one or more points along the edge of the patch, and the patch spans the open area. When the piston is activated to push the patch downward, the piston passes through the open area.

[0055] Specific embodiments of the annular retainer are described below. For example, FIG. 5A to FIG. 5C 1 is a perspective view, a top view, and a bottom view of an annular retainer 50 for a drug patch 10 according to an exemplary embodiment. The retainer has a rigid peripheral edge 58 and a central opening 56. Figure 5B and Figure 5C As shown, the patch is secured over opening 56 and secured inside edge 58. In some cases, patch 10 is retained by retaining ring 52 at one or more points along its edge.

[0056] As shown, the retainer 50 is circular, but the use of the term ring does not mean that the retainer must be circular. Depending on factors such as the shape and structure of the patch, the retainer can have other shapes, such as square, oval, triangle or other shapes.

[0057] A variety of connection mechanisms may be used to secure the annular retainer 50 to the base 120 of the actuator 122. For example, Figure 7 As shown, the retainer 50 and the actuator 122 may be connected by an extension 54 having a clip. Other connection mechanisms may be used, such as threaded connections, press fits, friction fits, or adhesive connections.

[0058] Variations in the ring holder configuration and patch retention methods were considered. For example, FIG. 5A to FIG. 5C A circular retaining ring 52 is shown, but the retaining ring may have other shapes, including a ring 52' ( 9A to 9C) to help remove and place on the holder. In addition, the retaining ring 52' can form a sandwich connection 57 (Figure 9c) with a flange and groove or similar structure to help stabilize the connection between the retaining ring 52' and the patch.

[0059] Furthermore, while the patch may be held in a holder that may be attached to the bottom of the actuator 122, other configurations are also contemplated. For example, a Figure 8 A small annular retainer 60 with a retaining ring 62 is shown, and such a retainer can be placed on top of the actuator, thereby avoiding the need for a larger annular retainer to be attached to the bottom of the actuator. In addition, instead of using a retaining ring, the patch 10 can be retained with a breakaway connector. For example, Fig.15 is an optional detachment mechanism 1400 for holding a drug patch in a patch applicator. In a detachment mechanism, a thin or relatively weak connector 1410 holds the patch on the applicator, and the connector 1410 breaks or tears due to the pressure of the piston.

[0060] A suitable patch would be designed to be released from the actuator by application of pressure from the piston 114 . Fig. 6A and Figure 6B 1 is an exemplary patch configuration for use with the disclosed applicator and patch system. As shown, the patch 10, 10' may include a backing layer 12, 12', an adhesive region 16, and a drug-containing region 14. The adhesive region 16 may extend into the drug-containing region 14 (e.g., a microneedle array) as long as the adhesive is placed in a manner that does not adversely affect the microneedles or their detachment from the skin.

[0061] The backing layer may extend from the periphery of the adhesive area 16 along the entire periphery, such as Fig. 6A as shown, or extending from a portion of the adhesive, such as at a corner, as Figure 6B As described above, at least a portion of the patch, such as the backing layer, may be sufficiently flexible to allow the patch to be pushed through the annular retainer of the applicator device 100. In order to provide sufficient flexibility, the backing layer can be formed of a material having mechanical properties and / or dimensions that provide the desired degree of flexibility. For example, a suitable material for the backing layer can be polyester (e.g., polyethylene terephthalate or polyethylene terephthalate approximately 0.002' or 0.005" thick), paper, aluminum, or other flexible material.

[0062] Furthermore, the patch 10, including the entire patch or the backing layer, can have a variety of shapes. For example, Fig.16A Figures C to C are alternative exemplary embodiments of the backing portion of the drug patch. The patch includes a polygon (e.g., a hexagon ( Fig.16A ) or octagon), square ( Fig. 16B ) or round ( Fig. 16C). In addition, other shapes are also considered, such as triangle, oval, square with rounded edges (circular or squarish), such as Fig. 6A or Figure 6B In addition, the backing layer can be modified to increase flexibility in certain areas. For example, Fig. 16C As shown, cutouts or notches 1500 may be provided in certain areas along the perimeter of the backing layer.

[0063] According to various embodiments, Fig.11A , Fig. 11B , Fig.12 13 and 14 provide further details regarding the structure and interaction of the actuator 122 and the piston 114. Fig.11A and Fig. 11B are perspective and side views illustrating the connection and movement of actuator 122 within an exemplary patch applicator device. Fig.12 yes Fig.11A and Fig. 11B 1 . A perspective view of the actuator shown in FIG. As shown, the actuator includes a base 128 and an extension 126. Typically, the actuator and the outer body 110 are engaged to allow only substantially linear sliding motion between the two. In this way, the extension 126 can engage the outer body through a groove, a tube, a sliding path, or a protrusion and a cam path type of connection. For example, the actuator 122 can have a protrusion 124 that engages the linear cam path 111 of the body 110. It is contemplated that this configuration can be changed, for example, by placing the protrusion on the body and the path on the actuator 122. In addition, although two extensions, protrusions, and cam paths are shown, three or more of each can be used.

[0064] As previously mentioned, the piston 114 can have a variety of configurations. Typically, the piston will be substantially cylindrical because its circular cross section will allow rotation within the body 110. Figure 13 is a perspective view of the piston 114 of an exemplary patch applicator device. As shown, the piston 114 has a protrusion 130, a top 132, and a flange or widened area 138, all of which have been discussed above.

[0065] The piston 114 has a bottom surface 136 configured to push the patch downwardly. As shown, the surface 136 is convex, but it is contemplated that the surface may be flat or have other modifications, such as a smooth or textured surface.

[0066] As described above in describing the movement of the piston and actuator, the actuator pushes the piston upward to compress the spring or compressible member, but once the piston reaches a certain point in the cam path, the piston is released, thereby releasing the energy stored in the now compressed spring and pushing the piston downward to apply the patch. It is contemplated that the piston and actuator can be engaged in different configurations to allow the piston to be released from the actuator. In one embodiment, the piston can rotate relative to the actuator within the outer body.

[0067] FIG. 14A to FIG. 14C 1 shows the interaction between the actuator 122 and the piston 114 of the applicator according to various embodiments. As shown, the actuator extension 126 pushes against the flange or widened portion 138 of the piston 114. However, the flange or widened portion 138 does not extend around the entire perimeter of the piston 114, but rather the widened portion 138 has a gap 139 ( Fig. 13B and Fig. 14C ). When the piston's protrusion 130 reaches the third downward portion 143 of the cam path 140, the actuator's extension 126 reaches the gap 139 of the piston's widened portion 138. This effect is due to the rotation of the piston 114 as the protrusion 130 moves through the annular cam path 140, as well as the linear movement of the actuator. Thus, the piston 114 is released downward as the engagement between the piston protrusion 130 and the cam path 140 and the engagement between the piston widened portion 138 and the actuator extension 126 are simultaneously released.

[0068] It should be noted that although the components including the piston 114, the actuator 122, and the outer body are described as having interacting protrusions 130 and 124 and cam paths 140 and cam paths 111, the locations of the protrusions and cam paths can be varied. For example, the cam path can be located on the actuator and / or the piston, have protrusions on the inner surface of the outer body 110, or a combination of these configurations.

[0069] Furthermore, while the annular retainer is attached to the bottom surface of the actuator, it is contemplated that the bottom surface of the actuator may be the skin contacting surface of the device and the pathway may be secured to the top surface of the actuator base 128 (eg, using retainer 60).

[0070] Detailed Example of an Exemplary Patch

[0071] As described above, the applicator can be used to apply a variety of types of drug patches, but may be particularly suitable for the application of microneedle devices. Accordingly, suitable patches including microneedles will be described in more detail below. It is contemplated that the applicator and / or annular holder or subassemblies can be provided as a kit or system including an applicator and a patch, a patch and annular holder, or an applicator with one or more patches for use with a reusable applicator. Suitable microneedle devices are further described in PCT patent application PCT / US2011 / 056856, entitled "Silk fibroin-based microneedles and methods of making the same," filed on October 19, 2011; PCT patent application PCT / US2019 / 025467, entitled "Microneedles comprising silk fibroin applied to a dissolvable substrate," filed on April 2, 2019; and PCT patent application PCT / US2020 / 055 filed on October 9, 2020. 139, entitled “Silk fibroin-based microneedles and their uses”; PCT application PCT / US2021 / 033776, filed on May 21, 2021, entitled “Compositions and devices for vaccine release and their uses”; PCT / US2022 / 030177, filed on May 20, 2022, entitled “Microneedle vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)”, each of which is incorporated herein by reference in its entirety.

[0072] Suitable patches may preferably include silk fibroin-based microneedles and microneedle devices (e.g., microneedle arrays and patches) for administration, delivery and release, e.g., controlled release or sustained release, of therapeutic agents, e.g., vaccines, antigens and / or immunogens (e.g., influenza vaccines and / or coronavirus vaccines, e.g., mRNA-based vaccines), across biological barriers, e.g., skin, mucosa, oral cavity, tissues or cell membranes.

[0073] The term "administering" or "dosing" includes a route for introducing a therapeutic agent into a subject to achieve its intended function. In some embodiments, the therapeutic agent can be administered repeatedly, such as by a microneedle or microneedle device as described herein, and the administration can be spaced apart by at least about 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 12 weeks, 2 months, 75 days, 3 months, or at least 6 months. In other embodiments, the therapeutic agent can be administered repeatedly every year, such as by a microneedle or microneedle device as described herein. In other embodiments, the therapeutic agent can be administered repeatedly as needed, such as by a microneedle or microneedle device as described herein, to achieve a therapeutic or preventive effect. Administration "in combination" with one or more other therapeutic agents includes simultaneous (concurrent) and continuous administration in any order.

[0074] As used herein, "subject" refers to a person or an animal. Typically, the animal is a vertebrate, such as a primate, a rodent, a livestock or a game animal. Primates include chimpanzees, crab-eating monkeys, spider monkeys and macaques (such as rhesus monkeys). Rodents include mice, rats, marmots, ferrets, rabbits and hamsters. Livestock and game animals include cattle, horses, pigs, deer, bison, buffalo, felines (such as house cats), canines (such as dogs, foxes, wolves), birds (such as chickens, emus, ostriches) and fish (such as trout, catfish and salmon). In some embodiments of 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. The mammal can be a human, a non-human primate, a mouse, a rat, a dog, a cat, a horse or a cow, but is not limited to these examples. In addition, the methods and formulations described herein can be used to treat domestic animals and / or pets. In some embodiments, the term "subject" is intended to include a living organism (eg, a mammal, eg, a human) in which an immune response can be elicited.

[0075] In certain embodiments, the subject is a human. The subject can be of any age. In one embodiment, the subject is an elderly human subject, e.g., 65 years of age or older. In one embodiment, the subject is a human subject who is not elderly, e.g., less than 65 years of age. In one embodiment, the subject is a human pediatric subject, e.g., 18 years of age or younger. In one embodiment, the subject is an adult subject, e.g., older than 18 years of age.

[0076] As used herein, the term "antigen" refers to a molecule (e.g., a gene product (e.g., a protein or peptide), a pathogen fragment, a complete pathogen, a viral vector, or a viral particle) that can induce a humoral immune response and / or a cellular immune response, for example, leading to the activation of B and / or T lymphocytes and / or innate immune cells and / or antigen presenting cells. Any macromolecule, including a protein or peptide, can be an antigen. Antigens can also be derived from genomes and / or recombinant DNA. For example, any DNA encoding an "antigen" comprising a nucleotide sequence or a partial nucleotide sequence encoding a protein that can elicit an immune response. In some embodiments, the antigen does not need to be encoded only by the full-length nucleotide sequence of the gene, and the antigen does not need to be encoded by the gene at all. In some embodiments, the antigen can be synthetic or can be derived from a biological sample, such as a tissue sample, a tumor sample, a cell, or a fluid with other biological components. In some embodiments, the antigen can be derived from a virus, such as an inactivated virus, a virus-like particle, or a viral vector. The antigen used herein can also be a mixture of several separate antigens.

[0077] As used herein, the term "immunogen" refers to any substance (e.g., antigen, antigen combination, pathogen fragment, complete pathogen) that can induce an immune response in an organism. "Immunogen" can induce an immune response against itself after being administered to a mammalian subject / administered to a mammalian subject. The term "immunology" used herein with respect to immune response refers to the development of humoral (antibody-mediated) and / or cellular (antigen-specific T cells or their secretory products mediated) responses to the immunogen in the recipient subject. This response can be an active response induced by inoculating the subject with an immunogen or immunogenic peptide, or a passive response induced by administering antibodies or sensitized T cells to the immunogen. In some embodiments, the immunogen is a coronavirus antigen. In some embodiments, the immunogen is a coronavirus. In some embodiments, the immunogen is an influenza virus. In some embodiments, the immunogen is a viral vaccine (e.g., a monovalent (also referred to as monovalent) or polyvalent (also referred to as polyvalent) vaccine, such as for coronavirus and / or influenza). In some embodiments, the vaccine (e.g., coronavirus vaccine and / or influenza vaccine) can be monovalent, bivalent, trivalent, tetravalent (also referred to as tetravalent) or pentavalent. In some embodiments, the immunogen is a replicating or non-replicating vaccine vector (e.g., including an adenovirus vector, an adeno-associated virus vector, an alphavirus vector, a herpes virus vector, a measles virus vector, a poxvirus vector, or a vesicular stomatitis virus vector).

[0078] As used herein, the terms "therapeutic agent" and "active agent" are art-recognized terms that refer to any chemical moiety that is a biologically, physiologically, or pharmacologically active substance that acts locally or systemically in a subject. Various forms of therapeutic agents can be used that are capable of being released from the microneedles described herein into adjacent tissues or fluids when administered to a subject. Examples of therapeutic agents, also known as "drugs," are described in well-known references such as The Merck Index, Physician's Desk Reference, and The Pharmacological Basis of Therapy, and include, but are not limited to, drugs; vitamins; mineral supplements; substances used to treat, prevent, diagnose, cure, or alleviate disease, such as viral infection; substances that affect the structure or function of the body; or prodrugs, which become biologically active or more active after being placed in a physiological environment.

[0079] In some embodiments, therapeutic agents include, but are not limited to, vaccines, antigens, and / or immunogens. In some embodiments, therapeutic agents include coronavirus vaccines, antigens, and / or immunogens. In some embodiments, therapeutic agents include influenza vaccines, antigens, and / or immunogens.

[0080] In some embodiments, therapeutic agents include, but are not limited to, amino acid molecules, such as peptides and / or proteins. In some embodiments, therapeutic agents include recombinant protein vaccines.

[0081] In some embodiments, therapeutic agents include but are not limited to nucleic acid molecules, such as deoxyribonucleic acid (DNA) molecules and / or ribonucleic acid (RNA) molecules. In specific embodiments, therapeutic agents include mRNA. In some embodiments, therapeutic agents include nucleic acid-based vaccines, such as DNA-based vaccines and / or RNA-based vaccines. In some embodiments, therapeutic agents include mRNA-based vaccines.

[0082] As used herein, the term "vaccine" refers to any composition that will induce a protective immune response in a subject exposed to the composition. The immune response may include the induction of antibodies and / or the induction of T cell responses. Typically, an "immune response" includes, but is not limited to, one or more of the following effects: the production or activation of antibodies, B cells, helper T cells, inhibitory T cells, and / or cytotoxic T cells, which are specific for one or more antigens contained in or derived from the composition or vaccine of interest. Preferably, the subject will exhibit a therapeutic or protective immune (memory) response, thereby enhancing resistance to new infections and / or reducing the clinical severity of the disease. This protection will be demonstrated by reducing the number or severity of one or more clinical symptoms associated with pathogen infection, or the lack of these symptoms, delaying the onset of viremia, reducing viral persistence, reducing total viral load, and / or reducing viral excretion. In some embodiments, "vaccine" refers to any preparation of an antigen or immunogen (including subunit antigens, toxoid antigens, binding antigens or other types of antigen molecules, or nucleic acid molecules encoding them) or inactivated or attenuated live microorganisms, which, when introduced into a subject, affects an immune response to a specific antigen or microorganism by causing activation of the immune system (e.g., inducing antibody formation, T cell response, and / or B cell response) against the specific antigen or microorganism. Typically, vaccines against microorganisms are directed against at least a portion of a virus, bacterium, parasite, mycoplasma, or other infectious agent.

[0083] The term "therapeutically effective amount" refers to an amount of a composition as defined herein that is effective in preventing, ameliorating and / or treating a condition resulting from a disease as described herein, such as a viral infection.

[0084] The term "treatment" refers to therapeutic treatment as well as prophylactic or preventive measures for curing or stopping or at least delaying the development of a disease. Patients in need of treatment include patients who already have a disease caused by a viral infection as described herein, as well as patients in need of prevention of a viral infection. Subjects who have partially or completely recovered from a viral infection as described herein may also need treatment. Prevention includes inhibiting or reducing the spread of a virus, or inhibiting or reducing the onset, development or progression of one or more symptoms associated with a viral infection as described herein.

[0085] As used herein, the term "virus" refers to an infectious agent consisting of nucleic acid wrapped in protein. This infectious agent cannot replicate autonomously (i.e., replication requires the use of the host cell's mechanism). The viral genome can be single-stranded (ss) or double-stranded (ds), RNA or DNA, and reverse transcriptase (RT) may or may not be used. In addition, ssRNA viruses can be positive (+) or negative (-). Exemplary viruses include, but are not limited to, dsDNA viruses (e.g., adenoviruses, herpes viruses, poxviruses), ssDNA viruses (e.g., parvoviruses), dsRNA viruses (e.g., reoviruses), (+) ssRNA viruses (e.g., picornaviruses, togaviruses, coronaviruses), (-) ssRNA viruses (e.g., orthomyxoviruses, rhabdoviruses), ssRNA-RT viruses, i.e., (+) positive RNA (e.g., retroviruses) and dsDNA viruses (hepadnaviruses) with intermediate DNA in the life cycle. In some embodiments, viruses may also include wild-type (natural) viruses, inactivated 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 (eg, human papillomavirus (HPV)), bacteriophages, recombinant viruses, and viral vectors. As used herein, the term "bacteriophage" refers to a virus that infects bacteria.

[0086] As used herein, the term "coronavirus" refers to a positive ssRNA virus in the Coronaviridae family. A coronavirus may be an alpha coronavirus, a beta coronavirus, a gamma coronavirus or a delta coronavirus. A coronavirus may be a live wild-type virus, a live attenuated virus, an inactivated virus (e.g., an ultraviolet inactivated virus), a chimeric virus or a recombinant virus. Coronaviruses are known to infect humans and other animals (e.g., birds and mammals). Examples of coronaviruses include severe acute respiratory syndrome coronavirus (SARS-CoV), severe acute respiratory syndrome virus 2 (SARS-CoV-2), Middle East respiratory syndrome coronavirus (MERS-CoV), human coronavirus 229E (HCoV-229E), human coronavirus NL63 (HCoV-NL63), human coronavirus OC43 (HCoV-OC43) and human coronavirus HKU1 (HCoV-HKLH).

[0087] As used herein, the term "influenza virus" refers to a negative-sense ssRNA virus in the family Orthomyxoviridae. The influenza virus may be a live wild-type virus, a live attenuated virus, an inactivated virus, a chimeric virus, or a recombinant virus. Examples of influenza viruses include influenza A, influenza B, influenza C, and influenza D.

[0088] The microneedles described herein can be any shape and / or geometry suitable for piercing a biological barrier, such as a skin layer, to achieve release of the vaccine in a subject, such as controlled release or sustained release. Non-limiting examples of the shape and / or geometry of the microneedles include: cylindrical, wedge-shaped, conical, pyramidal and / or irregular shapes, or any combination thereof.

[0089] As used herein, the terms "release" and "controlled or sustained release" refer to the release of a vaccine, antigen and / or immunogen (e.g., from a microneedle, microneedle device, formulation, composition, article, device and formulation described herein, e.g., from a fibroin-based microneedle tip described herein), such as a coronavirus vaccine, an influenza vaccine, or a combination thereof, for a period of time, e.g., 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 longer, e.g., from about 4 days to about 25 days, from about 10 days to about 20 days, from about 10 days to about 15 days, from about 12 days to about 16 days, e.g., from about 1 to 2 weeks, from about 1 to 3 weeks, or from about 1 to 4 weeks, e.g., from about 1 month to about 3 months). In some embodiments, controlled or sustained release of a vaccine, such as a coronavirus vaccine and / or an influenza vaccine, over a period of about 1 day to about 14 days, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days, by a microneedle, microneedle device, formulation, composition, article, device, or formulation described herein can result in, for example, broad-spectrum immunity of a subject. In some embodiments, vaccine formulations and formulations comprising silk fibroin have controlled or sustained release properties (e.g., formulated and / or configured to release the vaccine, for example, into the skin of a subject, over a period of time, or at least 1, 5, 10, 15, 30, or 45 minutes; over a period of time, or at least 1, 2, 3, 4, 5, 10, or 24 hours; over a period of time, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days; over a period of time, or at least 1, 2, 3, 4, 5, 6, 7, or 8 weeks; over a period of time, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 months; over a period of time, or at least 1, 2, 3, 4, or 5 years or longer).

[0090] In some embodiments, the microneedles of the present invention may include the following layers: (1) a backing material; (2) a dissolvable substrate; and (3) an implantable controlled-release or sustained-release tip. For example, the microneedles described herein may include a backing material applied to a dissolvable substrate that supports a distal controlled-release or sustained-release implantable tip comprising silk fibroin and a vaccine (e.g., a flu vaccine and / or a coronavirus vaccine, e.g., an mRNA-based vaccine, antigen, and / or immunogen).

[0091] As used herein, the term "backing" refers to a material suitable for bonding and / or adhering a microneedle assembly. In some embodiments, the backing material is suitable for bonding and / or adhering to a substrate (eg, a dissolvable substrate) of a microneedle described herein.

[0092] As used herein, the term "matrix" or "dissolvable substrate" refers to a layer that forms a microneedle base (e.g., used as a support for the distal microneedle tip (e.g., a fibroin tip) loaded with a vaccine, antigen and / or immunogen (e.g., a coronavirus vaccine, an influenza vaccine, or a combination 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%, or 90% or more of the matrix dissolves after application to a biological barrier, such as the skin, a mucosal surface, or the oral cavity.

[0093] The terms "sustained-release tip", "implantable sustained-release tip", "implantable microneedle tip" or "releasable tip", used interchangeably herein, refer to the distal end of a microneedle, such as a tip, which is capable of piercing a biological barrier, such as the skin, mucous surface or oral cavity of a subject, and depositing within the biological barrier, skin layer (e.g., dermis). In an embodiment, the tip comprises fibroin in an amount sufficient to maintain the release of a vaccine, such as a coronavirus vaccine (e.g., a SARS-CoV-2 vaccine) and / or an influenza vaccine for a long period of time, such as 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, such as about 4 days to about 30 days, about 5 days to about 25 days, about 10 days to about 20 days, about 10 days to about 15 days, about 4 days to about 14 days, about 14 days to about 15 days, for example, about 1 to 2 weeks, about 1 to 3 weeks, or about 1 to 4 weeks, for example, about 2 to 12 months). In some embodiments, the implantable slow-release tip comprises a coronavirus vaccine, antigen, and / or immunogen. In some embodiments, the implantable slow-release tip comprises a flu vaccine, antigen, and / or immunogen.

[0094] 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 vaccines, antigens, and / or immunogens across biological barriers, e.g., skin, tissues, or cell membranes. In some embodiments, the microneedle comprises a substrate (e.g., a dissolvable substrate as described herein), a tip (e.g., an implantable tip as described herein), and an optional backing material. In an embodiment, the height dimension of the microneedle is between about 350 μm and about 1500 μm (e.g., between about 350 μm and about 1500 μm, for example, 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, or about 1500 μm). In some embodiments, the microneedles are fabricated to have any size and / or geometry to enable the microneedle tip (e.g., a silk fibroin tip), such as an implantable sustained-release tip, to be deployed into the dermis of the skin at a depth between about 100 μm and about 900 μm (e.g., at a depth of about 800 μm) for release, such as controlled or sustained release of a vaccine (e.g., a coronavirus vaccine and / or a flu vaccine).

[0095] As used herein, the terms "microneedle patch" and "microneedle array" refer to a device comprising a plurality of microneedles, such as fibroin-based microneedles, such as microneedles arranged in a random or predetermined pattern, such as an array.

[0096] In some embodiments, the length of the microneedle may be between about 350 μm and 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 0 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, or about 1500 μm). In embodiments, the length of the microneedle can be made long enough to enable the delivery of an implantable tip containing a vaccine, antigen, and / or immunogen to the epidermis (e.g., about 10 μm to 120 μm below the surface of the skin) for controlled or sustained release as described herein, for example, to induce an immune response. In some embodiments, the length of the microneedle can be made long enough to enable the delivery of an implantable tip containing a vaccine, antigen, and / or immunogen to the dermis (e.g., about 60 μm to about 2.1 mm below the surface of the skin) for controlled or sustained release as described herein. One skilled in the art can adjust the microneedle length based on a variety of factors, including but not limited to tissue thickness, such as skin thickness (e.g., controlled or sustained release based on age, sex, location on the body, species (e.g., animal), drug delivery characteristics, diffusion characteristics of the vaccine, antigen, and / or immunogen (e.g., ionic charge and / or molecular weight, and / or shape of the vaccine, antigen, and / or immunogen)), or any combination thereof. However, without wishing to be bound by theory, using a microneedle of about 650 μm in height, an implantable sustained-release tip can be deployed to a subject at a depth of about 100 μm to about 600 μm within the dermis of the skin to achieve controlled or sustained release of the vaccine from the tip. In some embodiments, the microneedle can be about 800 μm in height (e.g., between about 500 μm and 1200 μm in height).

[0097] Exemplary microneedles of the present invention are FIG. 5A to FIG. 5B shown.

[0098] In some embodiments, a plurality of microneedles may be arranged in a random or predetermined pattern to form a microneedle array and / or patch, as described herein. The patch may include a carrier, backing, or "handling" layer adhered to the back of the substrate (see, e.g., FIG. 4 ). This layer may provide structural support and an area through which the patch may be handled and manipulated without disturbing the needle array.

[0099] Microneedle array

[0100] The microneedle array may include about 121 needles in an 11×11 square grid, with a spacing of about 0.75 mm. The individual needles are cones about 0.65 mm long, with a base diameter of about 0.35 mm and an included angle of about 30°. In order to penetrate the skin, the needle tips must be sharp. The radius of curvature of the tip is preferably no more than 0.01 mm.

[0101] Backing

[0102] Exemplary backing materials that can be used to make the microneedles of the present invention include, but are not limited to, solid phase carriers, such as paper-based materials, plastic materials, polymeric materials, or polyester-based materials (e.g., Whatman 903 paper, polymer tape, plastic tape, adhesive backing polyester tape, or other medical 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 backing polyester tape. In some embodiments, the backing material can be coated with (e.g., at least on one side) an adhesive suitable for bonding and / or adhering to a dissolvable substrate of the microneedles described herein.

[0103] The backing material used in the microneedle of the present invention can have a variety of characteristics, including but not limited to bonding and / or adhering to the ability of dissolving the base layer to allow demoulding. The backing material must be strong enough so that the backing keeps the integrity of the patch, for example, if the dissolving base layer has cracks or discontinuity. The backing material can have enough flexibility to adapt to, for example, uneven surfaces, such as skin surfaces. In particular, the backing must be soft enough during wearing, for example, after the patch is applied (for example, pressed into) the skin. The backing can include and / or be composed of a non-dissolving material so that the backing keeps its integrity after the patch is applied to the skin surface and during the patch is removed from the skin surface.

[0104] The backing can have any size suitable for the target skin surface. In some embodiments, the backing can be sized as a 12 mm diameter circle. In some embodiments, the backing can be sized as a 12 mm wide strip with a "handle" portion extending 12 mm beyond the edge of a 12 mm x 12 mm patch.

[0105] Dissolvable base

[0106] The dissolving substrate forms the base of the conical needle (e.g., used as a support for the distal silk core protein tip loaded with a vaccine, antigen and / or immunogen). The dissolving substrate can also be used as a layer connecting adjacent needles to form a microneedle array or patch. In some embodiments, the dissolving substrate contains 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 vaccine, antigen and / or immunogen loaded into the microneedle and / or microneedle device. In some embodiments, the dissolving substrate does not contain, for example, a detectable amount of vaccine, antigen and / or immunogen. In some embodiments, the dissolvable base layer is formulated to limit and / or reduce the amount of leakage (e.g., diffusion) of vaccines, antigens, and / or immunogens from the tips of the silk fibroin into the dissolvable base layer, for example, compared to base layer formulations known in the art, such as base layer formulations comprising PAA. In some embodiments, for example, the limit and / or reduction in leakage (e.g., diffusion) of vaccines, antigens, and / or immunogens from the tips of the silk fibroin can be determined at about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, or about 6 days after manufacture and storage (e.g., storage at about 4° C. (e.g., refrigeration), about 25° C. (e.g., room temperature), about 37° C. (e.g., body temperature), about 45° C. and / or about 50° C.); 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 more compared to base layer formulations comprising PAA.

[0107] The dissolvable base layer comprises a material that dissolves in the skin, for example, within the expected 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 dissolvable base layer dissolves within the expected 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 more) after application, for example, to the skin.

[0108] The material used to make the dissolvable substrate must be strong enough to allow the microneedles to penetrate the skin and tough enough (e.g., not brittle) to enable demolding. The dissolvable substrate material must withstand conventional handling without catastrophic failure and must maintain its mechanical properties between demolding and application (e.g., not hygroscopic to the point of melting due to ambient humidity). The dissolvable base material must be non-toxic and non-reactogenic at the dose used in the patch. In some embodiments, the dissolvable base comprises a water-soluble component. In some embodiments, as described herein, for example, the dissolvable base has improved biocompatibility compared to a dissolvable base comprising poly(acrylic acid) (PAA). In some embodiments, the dissolvable base material causes a reduced inflammatory response and / or reduced tissue necrosis. In some embodiments, the dissolvable base material is not PAA and induces a reduced inflammatory response and / or reduced tissue necrosis compared to PAA. In some embodiments, the dissolvable base material has a pH similar to the biological barrier into which it will dissolve, for example, a pH of about 4.0 to about 8.0.

[0109] Non-limiting examples of materials that can be used to make the dissolvable base layer include gelatin (e.g., hydrolyzed gelatin), polyethylene glycol (PEG), sucrose, low viscosity carboxymethyl cellulose (CMC), polyvinyl pyrrolidone (PVP), polyvinyl alcohol (PVA), hyaluronate, maltose and / or methyl cellulose. In some embodiments, the dissolvable substrate comprises one, two, three, four, five, six, seven, eight or more (e.g., all) of gelatin, polyethylene glycol (PEG), sucrose, carboxymethylcellulose (CMC), polyvinyl pyrrolidone (PVP), polyvinyl alcohol (PVA), hyaluronate, maltose, and methylcellulose, for example, at a concentration of between about 1% and about 75% (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 embodiments, the dissolvable substrate does not comprise a therapeutic agent, as described herein.

[0110] In some embodiments, the dissolvable substrate 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).

[0111] In some embodiments, the dissolvable substrate comprises about 1% to about 70% 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).

[0112] In some embodiments, the dissolvable base comprises 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).

[0113] In some embodiments, the dissolvable substrate comprises between about 1% and 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).

[0114] In some embodiments, the dissolvable base comprises about 10% to about 70% PVP (eg, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, or about 70% PVP).

[0115] In some embodiments, the dissolvable substrate comprises 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).

[0116] In some embodiments, the dissolvable base comprises from about 1% to about 75% (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% hyaluronate).

[0117] In some embodiments, the dissolvable base comprises between about 1% and about 75% (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).

[0118] In some embodiments, the dissolvable substrate comprises about 1% to about 75% (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).

[0119] In some embodiments, the dissolvable base layer may comprise 40% hydrolyzed gelatin, 10% sucrose w / v in deionized water. Optionally, the base layer may include 1% low viscosity carboxymethyl cellulose (CMC), which may reduce brittleness. In some embodiments, the dissolvable base layer may comprise polyvinyl pyrrolidone (PVP) at 10 kD MW, up to 50% w / v in deionized water; polyvinyl alcohol at 13 kD MW, up to 20% hydrolyzed 87% in deionized water; or CMC up to 10% in deionized water. The following combinations are also suitable for making the dissolvable base layer: 30% PVP and 10% PVA; 37% PVP, 5% PVA and 15% sucrose; or various other ratios of PVP, PVA and sucrose.

[0120] In some embodiments, the dissolvable substrate is approximately 12 mm square and 0.75 mm thick. In some embodiments, the dissolvable substrate can cover the entire patch. In some embodiments, the size of the substrate can be a 12 mm diameter circle, or a 12×12 mm square.

[0121] Implantable sustained-release tip

[0122] In an embodiment, the implantable slow-release tip may be made of silk fibroin and may contain a vaccine, antigen and / or immunogen described herein (e.g., a flu vaccine and / or a coronavirus vaccine, e.g., an mRNA-based vaccine). In some embodiments, the implantable slow-release tip may be designed to be deployed into the dermis of the skin (e.g., not deployed into the subcutaneous space) because the population of professional antigen-presenting cells in the dermis is much higher than that in the subcutaneous space. In humans, the thickness of the dermis ranges from about 1000 to 2000 μm (e.g., about 1 to 2 mm) based on location and patient age and health. In rodents, the dermis is much thinner (e.g., about 100 to 300 μm in mice and about 800 to 1200 μm in rats). Without wishing to be bound by theory, using 650 μm tall microneedles, implantable sustained-release tips can be deployed at a depth of between about 100 μm and about 600 μm to achieve controlled or sustained release of the vaccines, antigens and / or immunogens described herein (e.g., influenza vaccines and / or coronavirus vaccines, e.g., mRNA-based vaccines).

[0123] Without being bound by theory, the molecular weight of the silk fibroin solution used in the manufacture of microneedles described herein can be used as a controlling factor for regulating the controlled release or sustained release of vaccines, antigens and / or immunogens (e.g., influenza vaccines and / or coronavirus vaccines, e.g., vaccines based on mRNA) from the tip. In some embodiments, a higher molecular weight silk fibroin solution can be advantageous for a slower controlled release or sustained release (e.g., reducing the amount of the initial burst (e.g., the amount released on day 0) by at least about 10%, and then releasing additional antigens over at least about the next 4 days). In some embodiments, the controlled release or sustained release of vaccines, antigens and / or immunogens (e.g., influenza vaccines and / or coronavirus vaccines, e.g., vaccines based on mRNA) from the tip can last for 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 14 days, e.g., about 1 to 2 weeks, about 1 to 3 weeks or about 1 to 4 weeks). In some embodiments, the controlled release or sustained release occurs within about 1 week to about 2 weeks.

[0124] In an embodiment, the fibroin solution used to make the microneedles described herein may be a low molecular weight fibroin composition, which comprises a population of fibroin fragments having a certain molecular weight range, characterized in that no more than 15% of the total number of fibroin fragments in the population have a molecular weight greater than 200 kDa, and at least 50% of the total number of fibroin fragments in the population have a molecular weight within a specific range, wherein the specific range is between about 3.5 kDa and about 120 kDa, or between about 5 kDa and about 125 kDa. In other words, the fibroin solution used to make the microneedles described herein may comprise a population of fibroin fragments having a certain molecular weight range, characterized in that no more than 15% of the total molar number of fibroin fragments in the population have a molecular weight greater than 200 kDa, and at least 50% of the total molar number of fibroin fragments in the population have a molecular weight within a specific range, wherein the specific range is between about 3.5 kDa and about 120 kDa, or between about 5 kDa and about 125 kDa. (See, for example, WO 2014 / 145002, which is incorporated herein by reference).

[0125] Exemplary silk fibroin (e.g., regenerated silk fibroin) solutions may have different molecular weight distributions, as shown by size exclusion chromatography (SEC) determination (see, e.g., FIG. 5 ). In some embodiments, the silk fibroin solution may be prepared, for example, according to established methods. In some embodiments, prior to SEC analysis, the cocoon pieces from silkworms are first boiled in 0.02M Na2CO3 to remove sericin present in unprocessed natural silk. In some embodiments, the silk fibroin composition may be degummed from silkworm cocoons at atmospheric boiling temperatures for about 480 minutes or less, e.g., 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. In one embodiment, the fibroin composition can be prepared by degumming silk cocoons in an aqueous sodium carbonate solution at atmospheric boiling temperature for about 480 minutes or less, e.g., 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.

[0126] In some embodiments, the silk fibroin solution may be a 10 minute boil (10MB), 60 minute boil (60MB), 120 minute boil (120MB), 180 minute boil (180MB) or 480 minute boil (480MB) silk fibroin solution (see, e.g., FIG. 5 ). In some embodiments, the flu vaccine, antigen and / or immunogen may be formulated in a 10MB 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, the flu vaccine, antigen and / or immunogen may be formulated in a 60MB 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, influenza vaccines, antigens, and / or immunogens can be formulated in a 1% w / v to about 10% w / v (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% w / v) 120MB silk fibroin solution.

[0127] In some embodiments, the flu vaccine, antigen and / or immunogen can 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, the flu vaccine, antigen and / or immunogen can 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).

[0128] Without being bound by theory, the main tunability of the implantable sustained-release tip is its crystallinity, measured by the β-sheet content (inter- and intra-molecular β-sheets). This affects the solubility of the silk tip matrix and the ability of the antigen to be retained. As the β-sheet content increases, the mechanical strength of the tip also becomes higher. A specific vaccine release pattern is achieved by adjusting the crystallinity and diffusivity of the silk matrix. This is achieved by silk input materials and formulations and post-treatments to increase crystallinity (e.g., water annealing, methanol / solvent annealing). In some embodiments, the implantable controlled- or sustained-release microneedle tip contains about 10% to about 60% (e.g., about 10%, about 20%, about 30%, about 40%, about 50%, about 60%) of β-sheet content, for example based on a "crystallization index", such as a "crystallization index" known in the art. In some embodiments, the implantable controlled- or sustained-release microneedle tip can be formulated as particles (e.g., microparticles and / or nanoparticles).

[0129] Dimensions of implantable sustained-release tip

[0130] The methods provided herein can be used to make implantable sustained-release tips based on silk fibroin of any size, for example, having a height / length ranging from 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). In some embodiments, the implantable tip can have a diameter of any size, for example, based on the type of biological barrier (e.g., skin layer) that is intended to be pierced by the tip. In embodiments, the tip can have a size (e.g., diameter) ranging from 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 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 will be appreciated that there are no fundamental limitations that prevent sustained-release tips from having even smaller diameters (e.g., limitations of silk replica casting have been demonstrated with a resolution of tens of nanometers, see, e.g., Perry et al., 20 Adv. Mat. 3070 (2008)).

[0131] In certain embodiments, the sharpness of implantable sustained-release tip is described herein with tip radius.The mold for making microneedle described herein is designed to have a tip radius between about 0.5 μm to about 10 μm (for example, about 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 certain embodiments, the tip radius is between about 20 μm to about 25 μm (for example, about 20 μm, 21 μm, 22 μm, 23 μm, 24 μm or 25 μm). Not bound by theory, it is understood that a blunter needle may need greater force to penetrate the epidermis. In an embodiment, other sizes of implantable sustained-release tip can be controlled by the shape and filling volume of the mold. In some embodiments, the implantable sustained-release tip has an angle between about 5 degrees and 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 implantable sustained-release tip may have an angle between about 15 degrees and 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).

[0132] In an embodiment, the height of the implantable sustained-release tip may depend on the formulation and printing volume, which may affect surface tension and drying dynamics. In some embodiments, the height of the implantable sustained-release tip may extend to half of the full height of the microneedle. In some embodiments, the height of the implantable sustained-release tip is between about 75 μm and about 475 μm (e.g., about 75, 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, the base of the tip includes a thin "shell" layer (e.g., about 5, 6, 7, 8, 9 or 10 μm thick) with a thickness of about 5 to 10 μm. In some embodiments, the implantable sustained-release tip can be dried into a more solid configuration with a minimal "shell" where the height can be close to 150 μm (e.g., between about 50 μm and about 200 μm) and the thickness is >50 μm (e.g., between about 25 μm and about 75 μm).

[0133] In addition, the microneedles of the present invention can utilize known technologies that have been developed, for example, functionalizing silk fibroin (eg, active agents such as dyes and sensors). See, e.g., U.S. Patent No. 6,287,340, Bioengineered anterior cruciate ligament; WO 2004 / 000915, Silk biomaterials and methods of use thereof; WO 2004 / 001103, Silk biomaterials and methods of use thereof; WO 2004 / 062697, Silk fibroin materials and uses thereof; WO 2005 / 000483, Methods of forming inorganic coatings; WO 2005 / 012606, Concentrated aqueous silk fibroin solutions and uses thereof; WO 20111005381, Vortex-induced gelation of silk fibroin for encapsulation and delivery; WO 20051123114, Silk-based drug delivery systems; WO 2006 / 076711, Fibrous protein fusions and their use in the formation of advanced organic / inorganic composites; U.S. Appl. Publisher No. 2007 / 0212730, Covalently immobilized protein gradients in three-dimensional porous holders; WO 2006 / 042287, Method for producing biomaterial holders; WO2007 / 016524, Method for gradually depositing silk fibroin coatings; WO 2008 / 085904, Biodegradable electronic devices; WO20081118133, Silk microspheres for encapsulation and controlled release; WO 20081108838, Microfluidic devices and methods for making the same; WO 20081127404, Nanopatterned biopolymer devices and methods for making the same; WO 20081118211, Biopolymer photonic crystals and methods for making the same; WO 20081127402, Biopolymer sensors and methods for making the same; WO20081127403, Biopolymer optofluidic devices and methods for making the same; WO 20081127401, Biopolymer optical waveguides and methods for making the same; WO 20081140562, Biopolymer sensors and methods for making the same; WO 20081127405, Microfluidic device with cylindrical microchannel and method for manufacturing the same; WO 20081106485, Tissue engineered silk organ; WO20081140562, Electroactive biopolymer optical and electro-optical devices and methods for manufacturing the same; WO 20081150861, Method for gelation of silk fibroin using ultrasound treatment; WO 20071103442, Biocompatible holder and adipose-derived stem cells; WO 20091155397, Edible holographic silk products; WO 20091100280, Three-dimensional silk hydroxyapatite composition; WO2009 / 061823, Fabrication of silk fibroin photonic structures by nanocontact imprinting; WO 20091126689, Systems and methods for fabricating biomaterial structures.

[0134] In various embodiments, the fibroin-based microneedle tip may further comprise at least one additional therapeutic agent, wherein the additional therapeutic agent may be dispersed throughout the microneedle or form at least a portion of the microneedle tip. In some embodiments, the additional therapeutic agent may be used to treat viral infections as described herein. As described herein, optionally, the fibroin-based microneedle tip may further comprise an excipient and / or an adjuvant.

[0135] Viruses, Antigens, and Immunogens

[0136] In some embodiments, the present invention provides various therapeutic agents, for example, vaccines, antigens and / or immunogens derived from viruses that are members of the Orthomyxovirus family, for example, by formulations, compositions, articles, devices, formulations, microneedles and / or microneedle devices (e.g., microneedle patches) described herein and / or according to methods described herein, for example, controlled or sustained delivery. In some embodiments, the vaccines, microneedles and / or microneedle devices (e.g., microneedle patches) described herein may comprise negative sense ssRNA viruses and / or RNA viruses, such as influenza viruses. In some embodiments, vaccines, antigens and / or immunogens include nucleic acids (e.g., DNA and / or RNA) derived from influenza viruses. In some embodiments, vaccines, antigens and / or immunogens comprise amino acids (e.g., peptides and / or proteins) derived from influenza viruses. In some embodiments, influenza vaccines, antigens and / or immunogens comprise inactivated and / or attenuated live vaccines or split vaccines of influenza viruses. In some embodiments, vaccines and / or microneedles comprise non-replicating viral antigens.

[0137] Specifically, the present invention contemplates vaccines, microneedles and / or microneedle devices (e.g., microneedle patches) comprising influenza virus vaccines, antigens and / or immunogens. Influenza viruses are RNA viruses (e.g., linear negative sense single-stranded RNA viruses). There are four known influenza virus genera, each containing a type (e.g., influenza A, B, C, and D). Influenza viruses can constantly change and are affected by antigenic drift and antigenic shift. Exemplary influenza strains are further described in the Examples (see, e.g., Tables 1 and 2).

[0138] Influenza A can be divided into subtypes according to two proteins on the surface of the virus: hemagglutinin (HA) and neuraminidase (NA). Influenza A includes 18 known HA subtypes, referred to herein as H1 to H18, and 11 known NA subtypes, referred to herein as N1 to N11. Many different combinations of HA and NA proteins can be found on the surface of influenza A viruses. For example, "H1N1 virus" refers to an influenza A virus subtype comprising an H1 protein and an N1 protein. Exemplary influenza A virus subtypes confirmed to infect humans include, but are not limited to, H1N1, H3N2, H2N2, H5N1, H7N7, H1N2, H9N2, H7N2, H7N3, H10N7, and H7N9. H1N1 virus and H3N2 virus are currently widely spread among the population.

[0139] Exemplary influenza B viruses can belong to, for example, the B / Yamagata lineage and / or the B / Victoria lineage.

[0140] vaccine

[0141] Non-limiting examples of influenza vaccines for the microneedles and microneedle devices (such as microneedle patches) described herein may include commercially available vaccines, such as seasonal vaccines, pandemic vaccines, and / or universal vaccines; egg-based vaccines, cell culture-based vaccines; recombinant vaccines; live attenuated, inactivated whole viruses, split vaccines, and / or protein subunit vaccines; and adjuvanted vaccines. Various commercial influenza vaccines are listed below. In addition, influenza vaccines containing mRNA, DNA, viral vectors, and / or virus-like particles (VLPs) are suitable for the microneedles and microneedle devices (such as microneedle patches) described herein. In some embodiments, the influenza vaccine can target matrix protein 1, matrix protein 2 (M2e), and / or nucleoprotein (NP) of influenza virus.

[0142]

[0143]

[0144] Table 1. Exemplary vaccines

[0145] Vaccine formulations and compositions for controlled or sustained release

[0146] At least one vaccine, antigen and / or immunogen described herein (e.g., at least one vaccine, antigen and / or immunogen derived from an influenza virus described herein) can be incorporated into various preparations, compositions, products, devices and / or preparations for administration, for example, to achieve controlled release and / or sustained release. More specifically, at least one vaccine, antigen and / or immunogen described herein (e.g., at least one vaccine, antigen and / or immunogen derived from an influenza virus described herein) can be formulated into a preparation, composition, product, device and / or preparation by combining with a suitable pharmaceutically acceptable carrier or diluent, and can be formulated into a preparation in semi-solid, solid or liquid form. In some embodiments, the preparations, compositions, products, devices and / or preparations described herein contain silk fibroin. Exemplary preparations, compositions, products, devices and / or preparations include: microneedles (e.g., microneedle devices, e.g., microneedle patches as described herein), implantable devices (e.g., pumps, e.g., subcutaneous pumps), injectable preparations, reservoirs, gels (e.g., hydrogels), implants and particles (e.g., microparticles and / or nanoparticles). Thus, administration of the composition can be achieved in a variety of ways, including intradermal, intramuscular, transdermal, subcutaneous or intravenous administration. In addition, formulations, compositions, products, devices and / or preparations can be formulated and / or administered to achieve controlled and / or sustained release of at least one vaccine, antigen and / or immunogen described herein (e.g., at least one vaccine, antigen and / or immunogen derived from influenza virus described herein).

[0147] In some embodiments, the vaccine (e.g., flu vaccine) is administered for a period of time, or at least 1, 5, 10, 15, 30, or 45 minutes; for a period of time, or at least 1, 2, 3, 4, 5, 10, or 24 hours; for a period of time, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days; for a period of time, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days; for a period of time, or at least 1, 2, 3, 4, 5, 6, 7, or 8 weeks; for a period of time, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 months; for a period of time, or to 1, 2, 3, 4, or 5 years or longer, for example, substantially continuously. In one embodiment, the vaccine (e.g., flu vaccine) is administered as a controlled release or sustained release formulation, dosage form, or device. In some embodiments, the vaccine (e.g., flu vaccine) 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 vaccine 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 vaccine (e.g., influenza vaccine) is a silk-based controlled or sustained release dosage form or formulation (e.g., microneedles as described herein). In one embodiment, the vaccine (e.g., influenza vaccine) is administered by an implantable device, such as a pump (e.g., subcutaneous pump), an implant, an implanted tip of a microneedle, or a reservoir. The delivery method can be optimized so that a vaccine described herein (e.g., a flu vaccine and / or a coronavirus vaccine, e.g., an mRNA-based vaccine) dose (e.g., a standard dose) is administered and / or maintained in a subject for a predetermined time (e.g., a period of time, or at least: 1, 5, 10, 15, 30, or 45 minutes; 1, 2, 3, 4, 5, 10, or 24 hours; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days; 1, 2, 3, 4, 5, 6, 7, or 8 weeks; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 months; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 months; 1, 2, 3, 4, or 5 years or longer). A substantially sustained or extended release of a vaccine (e.g., a flu vaccine) can be used to prevent or treat a viral infection (e.g., an influenza virus infection) for hours, days, weeks, months, or years.

[0148] In some embodiments, the present invention provides formulations, compositions, articles, devices and / or preparations of the present invention, which can be formulated and / or configured for controlled or sustained release of at least one vaccine, antigen and / or immunogen (e.g., at least one vaccine, antigen and / or immunogen derived from an influenza virus described herein) in an amount (e.g., a dose) sufficient to cause an immune response (e.g., a cellular immune response and / or a humoral immune response) against a virus, such as an influenza virus, in a subject and / or over a period of time.

[0149] In some embodiments, the formulations, compositions, articles, devices and / or preparations of the invention may be formulated and / or configured for controlled or sustained release of at least one vaccine, antigen and / or immunogen (e.g., at least one vaccine, antigen and / or immunogen derived from an influenza virus described herein) in an amount (e.g., dose) sufficient to produce broad-spectrum immunity in a subject and / or over a period of time.

[0150] Substantially continuous or extended release delivery or formulation of a vaccine (eg, an influenza vaccine) can be used to prevent or treat a viral infection (eg, an influenza virus infection) for hours, days, weeks, months, or years.

[0151] In some embodiments, at least one vaccine, antigen and / or immunogen described herein may be added to the silk fibroin solution, for example, before forming the silk fibroin microneedles or microneedle devices described herein. In embodiments, the silk fibroin solution may be mixed with the vaccine, antigen and / or immunogen and then used to make an implantable microneedle tip, for example, by a process of filling and / or casting, drying and / or annealing to make microneedles with any desired material properties, as described herein.

[0152] Without being bound by theory, the ratio of silk fibroin to vaccine, antigen and / or immunogen in the implantable tip of the microneedle affects its release. In some embodiments, increased silk concentration in the implantable tip facilitates slower release and / or greater antigen retention in the tip. Any concentration of silk can be used as long as the concentration allows printing and has sufficient mechanical strength to pierce the skin.

[0153] In some embodiments, fibroin can be used to make microneedles or components thereof, as described herein, 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).

[0154] Exemplary Excipients

[0155] In addition, the preparations, compositions, articles, devices and / or formulations can be formulated with common excipients, diluents or carriers for administration by intradermal, intramuscular, transdermal, subcutaneous or intravenous routes. In some embodiments, the preparations, compositions, articles, devices and / or formulations can be administered, for example, transdermally, and can be formulated into controlled release or sustained release dosage forms, etc. The preparations, compositions, articles, devices and / or formulations described herein can be administered alone, in combination with each other, or they can be used in combination with other known therapeutic agents.

[0156] Formulations suitable for the present invention are found in Remington's Pharmaceutical Sciences (1985). In addition, for a review of drug delivery methods, see Langer (1990) Science 249: 1527-1533. The formulations, compositions, articles, devices and / or preparations described herein can be manufactured in a manner known to those skilled in the art, for example, by mixing, dissolving, granulating, making dragees, grinding, emulsifying, encapsulating, embedding or lyophilizing processes. The following methods and excipients are merely exemplary and not restrictive.

[0157] The silk fibroin preparation used to make the microneedles described herein may include an excipient. In an embodiment, the excipient may be included to improve the stability of the incorporated vaccine, antigen and / or immunogen; increase the porosity of the silk matrix and the diffusion of the vaccine, antigen and / or immunogen from the preparation, composition, article, device, preparation and / or microneedle, such as the tip of the microneedle; and / or increase the crystallinity / β-sheet content of the silk matrix to make the silk material insoluble.

[0158] Exemplary excipients include, but are not limited to, sugars or sugar alcohols (e.g., sucrose, trehalose, sorbitol, mannitol, or a combination thereof), divalent cations (e.g., Ca2+, Mg2+, Mn2+, and Cu2+), and / or buffers. In some embodiments, the concentration of the excipient can be used to change the porosity of the matrix, for example, sucrose is used as the most common excipient for this purpose. Excipients can also be added to promote the self-assembly of silk into an ordered β-folded secondary structure, and such excipients can generally participate in hydrogen bonding or interact with the charge of the silk to achieve this effect. Non-limiting examples of excipients that can be used to promote the self-assembly of silk into an ordered β-folded secondary structure include monosodium glutamate (e.g., L-glutamic acid), lysine, sugar alcohols (e.g., sorbitol and / or glycerol), and solvents (e.g., DMSO, methanol, and / or ethanol).

[0159] In some embodiments, the sugar or sugar alcohol is sucrose, which is present in an amount 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, before drying.

[0160] In some embodiments, the sugar or sugar alcohol is sucrose, which is present in an amount 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), e.g., prior to drying.

[0161] In some embodiments, the sugar or sugar alcohol is trehalose, which is present in an amount 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, before drying.

[0162] In some embodiments, the sugar or sugar alcohol is sorbitol, which is present in an amount 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), e.g., prior to drying.

[0163] In some embodiments, the sugar or sugar alcohol is glycerol, which is present in an amount 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), e.g., prior to drying.

[0164] In some embodiments, the vaccine formulation further comprises a divalent cation. In some embodiments, the divalent cation is selected from the group consisting of Ca2+, Mg2+, Mn2+, and Cu2+. In some embodiments, the divalent cation is present in the formulation in an amount between 0.1 mM and 100 mM, for example, before drying. In some embodiments, the divalent cation is present in an amount of 10 mM per standard dose of the viral immunogen. -7 Up to 10 -4 The amount of divalent cations present in the formulation is molar, for example, immediately prior to drying. In some embodiments, the divalent cations are present in the formulation immediately prior to drying at a concentration of 10 -10 Up to 2×10 -3 The molar amount present in the formulation.

[0165] In some embodiments, the vaccine formulation further comprises poly(lactic-co-glycolic acid) (PGLA).

[0166] In some embodiments, the viral vaccine formulation further comprises a buffer, e.g., just before drying. In some embodiments, the buffer has a buffering capacity between pH 3 and pH 8, between pH 4 and pH 7.5, or between pH 5 and pH 7. In some embodiments, the buffer is selected from HEPES and CP buffers. In some embodiments, the buffer is present in the formulation in an amount between 0.1 mM and 100 mM, e.g., just before drying. In some embodiments, the buffer is present in an amount of 10 mM per standard dose of viral immunogen. -7 Up to 10 -4 In some embodiments, the buffer is present in an amount of 10 -10 Up to 2×10 -3 Exists in amounts between moles.

[0167] In addition, the vaccine can also be formulated into a reservoir, gel or hydrogel formulation. Such long-acting preparations can be administered by implantation (e.g., subcutaneous or intramuscular) or intramuscular injection. Thus, for example, the vaccine can be formulated with a suitable polymeric or hydrophobic material (e.g., as an emulsion in an acceptable oil) or an ion exchange resin, or as a sparingly soluble derivative, e.g., as a sparingly soluble salt.

[0168] In one embodiment, vaccination is performed by an implantable infusion device, such as a pump (e.g., a subcutaneous pump), an implant, or a reservoir. An implantable infusion device typically includes a housing comprising a reservoir that can be percutaneously filled with a subcutaneous injection needle that penetrates a filling port diaphragm. A drug reservoir is typically connected to a device outlet via an internal flow path for delivery of liquid to a patient's body part via a catheter. A typical infusion device also includes a controller and a fluid transfer mechanism, such as a pump or a valve, for moving liquid from the reservoir to the outlet of the device via an internal flow path.

[0169] In certain embodiments, the vaccine can be packaged and / or formulated into particles, such as microparticles and / or nanoparticles. Typically, the diameter of the nanoparticle is 10, 15, 20, 25, 30, 35, 45, 50, 75, 100, 150 or 200nm or 200 to 1000nm, such as 10, 15, 20, 25, 30, 35, 45, 50, 75, 100, 150 or 200nm or 20 or 30 or 50 to 400nm. Smaller particles can often be removed from the system faster. Therapeutic agents including vaccines can be embedded or coupled, such as covalently coupled or otherwise adhered to the nanoparticles.

[0170] Lipid or oil-based nanoparticles, such as liposomes and solid lipid nanoparticles, can be used to deliver therapeutic agents described herein, such as vaccines. Serpe et al., (2004) Eur. J. Pharm. Biopharm. 58: 673-680 and Lu et al., (20060 Eur. J. Pharm. Sci. 28: 86-95 describe solid lipid nanoparticles for delivering therapeutic agents. Polymer-based nanoparticles, such as PLGA-based nanoparticles, can be used to deliver the agents described herein. These tend to rely on a biodegradable skeleton in which the therapeutic agent is embedded (covalently linked to the polymer or not) in a polymer matrix. PLGA is widely used in polymer nanoparticles, see 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 Danhhier et al., (2009) J. Control. Release 133:11-17, Gryparis et al., (2007) Eur. J. Pharm. Biopharm. 67:1-8. Metal-based nanoparticles, such as gold-based nanoparticles, can also be used to deliver therapeutic agents. Protein-based, such as albumin-based nanoparticles can be used to deliver the agents described herein. In some embodiments, the therapeutic agent can be bound to nanoparticles of human albumin.

[0171] A variety of nanoparticles are known in the art. Exemplary methods include those described in WO2010 / 005726, WO2010 / 005723, WO2010 / 005721, WO2010 / 121949, WO2010 / 0075072, WO2010 / 068866, WO2010 / 005740, WG2006 / 014626, 7,820,788, 7,780,984, the contents of which are incorporated herein by reference in their entirety.

[0172] dose

[0173] According to the methods described herein, any dose (e.g., standard dose and / or split dose) of a vaccine, antigen and / or immunogen that is capable of eliciting an immune response (e.g., immunogenicity and / or broad-spectrum immunity) in a subject can be used, for example, when administered by the microneedles of the present invention. In some embodiments, a dose, such as a standard dose (e.g., human dose) of a vaccine, antigen and / or immunogen (e.g., a flu vaccine and / or a coronavirus vaccine, such as an mRNA-based vaccine) is between about 0.1 μg and about 65 μg (e.g., between about 0.1 μg and about 10 μg, between about 0.1 μg and about 1 μg, between about 0.5 μg and about 5 μg, between about 5 μg and about 10 μg, between about 10 μg and about 20 μg, between about 20 μg and about 30 μg, between about 30 μg and about 40 μg, between about 40 μg and about 50 μg, between about 50 μg and about 65 μg, for example, about 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, or 65 μg). In some embodiments, the dosage of a vaccine described herein (e.g., an influenza vaccine and / or a coronavirus vaccine, e.g., an mRNA-based vaccine), e.g., a standard human dose, is about 1 μg to about 30 μg per strain, e.g., about 5 μg to about 30 μg per strain (e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 μg per strain). In some embodiments, the dose of a vaccine described herein (e.g., an influenza vaccine and / or a coronavirus vaccine, e.g., an mRNA-based vaccine), e.g., a divided dose, does not exceed 1 / X of the total dose (e.g., a standard dose), 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. It is known in the art that there is a clinical precedent for sparing doses when influenza vaccines are delivered to the intradermal space (e.g., Fluzone ID), and the dose is about 9 μg per strain.Thus, in some embodiments, the total dose of influenza vaccine (e.g., Fluzone ID) that can be delivered by the microneedles of the present invention can be between about 5 μg and 13 μg (e.g., about 5 μg, about 6 μg, about 7 μg, about 8 μg, about 9 μg, about 10 μg, about 11 μg, about 12 μg, or about 13 μg).

[0174] Without wishing to be bound by theory, the total dose (e.g., a standard dose) of a vaccine, antigen, and / or immunogen administered by microneedles described herein can be distributed among a plurality of microneedles (e.g., within a patch) such that the microneedle tips can contain less than about 1% of the total dose (e.g., in an array comprising 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. In some embodiments, the implantable microneedle tip may contain about 0.1 μg to about 65 μg (e.g., about 0.1 μg, about 0.2 μg, about 0.3 μg, about 0.4 μg, about 0.5 μg, about 0.6 μg, about 0.7 μg, about 0.8 μg, about 0.9 μg, about 1 μg, about 1 μg to about 10 μg, about 10 μg to about 20 μg, about 20 μg to about 30 μg, about 30 μg to about 40 μg, about 40 μg to about 50 μg, or about 50 μg to about 65 μg) of a vaccine, antigen, and / or immunogen as described herein.

[0175] In some embodiments, the vaccine dose loaded into the microneedle patch can be controlled by the antigen concentration in the formulation solution forming the needle tip, the volume of solution distributed to each needle tip, and the total number of needles (the first two are more convenient methods for changing the dose). The dose released into the skin is related to the deployment efficiency (the portion of the needle tip remaining in the skin after removing the patch), and is also related to the release curve over time and the residence time of the tip in the skin. As the skin is constantly shed from the epidermis, deeper deployment in the skin is related to longer residence time. Therefore, it is desirable to maximize the penetration depth of the needle tip (reaching the limit defined by the depth of the pain receptors in the skin, for example, a depth between about 100 μm and about 600 μm), and it is also desirable that the antigen is spatially concentrated toward the needle tip.

[0176] The preparations, compositions, articles, devices and / or preparations described herein, including implantable sustained-release tip preparations, are designed to continuously release vaccine antigens not only during the duration that the tip is retained in the dermis, for example, but also during this period (e.g., at least about 1 to 2 weeks) to maintain the stability of the antigen. In some embodiments, it can be expected that about 95 to 100% of the total dose incorporated in the preparations, compositions, articles, devices, preparations and / or microneedles described herein can be used for delivery to, for example, a subject, for example, delivered to a subject's tissue, such as skin, mucosa, organ tissue, oral cavity, tissue or cell membrane. Without being bound by theory, the successful deployment of microneedles in the skin is at least about 50% of the array, and can be as high as 100% (e.g., at least about 50%, 60%, 70%, 80%, 90% or more (e.g., 100%) of the total number of microneedles constituting the array are successfully deployed, for example, in the skin for controlled or sustained release of vaccine antigens). In some embodiments, during deployment, a portion of the antigen may not be released from the silk tip.

[0177] use

[0178] The present invention also provides a method for delivering vaccines, antigens and / or immunogens (e.g., influenza vaccines and / or coronavirus vaccines, e.g., mRNA-based vaccines) across a biological barrier (e.g., skin). Such a method may include providing a formulation, composition, article, device, formulation and / or microneedle as described herein. For example, such a method may include providing at least one microneedle or at least one microneedle device as described herein, wherein the microneedle or microneedle device comprises a fibroin-based implantable tip having at least one vaccine, antigen and / or immunogen (e.g., influenza vaccine and / or coronavirus vaccine, e.g., mRNA-based vaccine); causing the microneedle or microneedle device to penetrate a biological barrier (e.g., skin); and causing the vaccine, antigen and / or immunogen to be released from the implantable tip over a period of at least about 4 days (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 14 days, e.g., about 1 to 2 weeks, about 1 to 3 weeks, or about 1 to 4 weeks). In some embodiments, the vaccine, antigen and / or immunogen is released into the biological barrier by degradation and / or dissolution of the implantable microneedle tip. In some embodiments, the microneedle or microneedle device is configured to vaccinate, antigen and / or immunogen in an amount and / or duration to produce broad-spectrum immunity in the subject, such as immunity to one or more viral antigens not present in the implantable slow-release tip, such as immunity to drift strains not present in the implantable slow-release tip.

[0179] The present invention also provides a method for providing broad-spectrum immunity to a virus, for example, influenza virus, in a subject, the method comprising vaccinating (e.g., influenza vaccine and / or coronavirus vaccine, for example, mRNA-based vaccine) in an amount (e.g., dosage) and / or time period sufficient to produce broad-spectrum immunity to the virus, for example, to produce an immune response (e.g., cellular immune response and / or humoral immune response) to a drift strain of the virus. In some embodiments, the vaccine is inoculated in the form of a composition for controlled or sustained release of the vaccine (e.g., for controlled or sustained release of one or more viral antigens described herein). In some embodiments, the vaccine is inoculated by a device for controlled or sustained release of the vaccine (e.g., for controlled or sustained release of one or more viral antigens described herein). The vaccine can be inoculated into a subject, for example, into a tissue or cavity selected from the skin, mucous membrane, organ tissue, muscle tissue, or oral cavity of the subject.

[0180] In some embodiments, the methods described herein include administering in an amount (e.g., a dose) and / or for a period of time sufficient to result in one or more of the following: (i) exposing a subject to one or more antigens in a vaccine in an amount and / or for a period of time sufficient to result in broad immunity in the subject, e.g., to result in an immune response (e.g., a cellular immune response and / or a humoral immune response) against a drifted strain of the virus; or (ii) a level of one or more antigens in a subject that is substantially stable, e.g., about 20%, 15%, 10%, 5% or 1%, relative to the amount required to result in an immune response (e.g., a cellular immune response and / or a humoral immune response) to the one or more antigens, e.g., a minimum amount. In some embodiments, the composition or device for controlled or sustained release of the vaccine is selected from: a microneedle (e.g., a microneedle device, e.g., a microneedle patch, e.g., as described herein), an implantable device (e.g., a pump, e.g., a subcutaneous pump), an injectable formulation, a reservoir, a gel (e.g., a hydrogel), an implant, or a particle (e.g., a microparticle and / or a nanoparticle).

[0181] In some embodiments, vaccination, for example, is released to, for example, a subject by a composition or device for controlled release or sustained release of a vaccine, to maintain a vaccine dose (e.g., antigen concentration) in a time period sufficient to produce broad-spectrum immunity in the subject, for example, to produce an immune response (e.g., cellular immune response and / or humoral immune response) to a viral drift strain (e.g., wherein the time period is about 1 to 21 days, for example, about 5 to 10 days or about 5 to 7 days, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days). Compositions or devices for controlled or sustained release of vaccines can maintain antigen release and / or levels in a subject over a sustained period of time. In some embodiments, compositions or devices for controlled or sustained release of vaccines maintain continuous or discontinuous release of antigens to a subject over a sustained period of time. Vaccine can be inoculated in the time period including at least about one week, for example, about 1 to 2 weeks, about 1 to 3 weeks or about 1 to 4 weeks, for example, by the composition or device for controlled release or sustained release. In certain embodiments, in a period of time including at least about 4 days (for example, about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 days or more days, for example, about 4 days to about 2 weeks, about 4 days to about 1 week), vaccination, for example, by the composition or device for controlled release or sustained release release vaccine.

[0182] The vaccine can be administered at a dose of about 0.1 μg to about 65 μg per strain, for example, about 0.2 μg to about 50 μg per strain (e.g., about 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, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65 1, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64 or 65 μg). In some embodiments, at least about 1% of the vaccine dose (e.g., at least about 0.5% to about 10%, at least about 5% to about 15%, at least about 10% to about 20% of the dose) is maintained, e.g., released by a composition or device for controlled or sustained release of a vaccine, e.g., into a subject, over a period of time comprising at least about 4 days (e.g., about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days, or more days, e.g., between about 4 days to about 2 weeks, between about 4 days to about 1 week).

[0183] In some embodiments, the vaccine is administered as multiple sub-doses of a total dose (e.g., a standard dose) over a period of time, for example, the vaccine is released by a composition or device for controlled or sustained release, for example, to achieve an immune response and / or broad-spectrum immunity, wherein the amount of vaccine administered in each sub-dose does not exceed 1 / X, wherein X is any number, for example, wherein 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 of the total dose of the vaccine (e.g., a standard dose).

[0184] In some embodiments, the vaccine is administered, for example, by a composition or device for controlled or sustained release of the vaccine, into, for example, the skin of a subject, in multiple doses corresponding to a percentage of the total dose (e.g., a percentage of a standard dose) over a period of time, for example, to achieve broad spectrum immunity, wherein the amount of vaccine administered in each of the multiple doses is about X%, where X is any number, for example, 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, 100, 125, 150, 175, 200, 300, 400, or 500 or more of the total dose of the vaccine (e.g., a standard dose).

[0185] Vaccination can be performed according to any of the methods described herein to achieve broad immunity, for example, to achieve an immune response against drifted strains, for example, a cellular and / or humoral immune response.

[0186] Without wishing to be bound by theory, a subject exposed to and / or infected with a first influenza virus may produce an immune response (e.g., a cellular and / or humoral immune response) resulting in the production of antibodies against the first influenza virus. Over time, antigenic changes (e.g., mutations) in the first influenza virus accumulate, and the antibodies produced by the subject against the first influenza virus may no longer recognize drift viruses (e.g., antigenically different strains). Using the methods, dosing regimens, microneedles, and microneedle devices described herein, it is possible to confer broad-spectrum immunity to subjects exposed to, infected with, and / or at risk of influenza infection. In addition, using the methods, dosing regimens, microneedles, and microneedle devices described herein, for example, compared to traditional burst release administration of vaccines, improved immunogenicity and / or broad-spectrum immunity can be conferred to subjects. For example, the improvement in immunogenicity and / or broad-spectrum immunity detectable in a subject can be greater (e.g., 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, or 15-fold or greater) compared to traditional burst-release administration of a vaccine, e.g., single-dose administration or bolus administration of a vaccine.

[0187] In some embodiments, the implantable sustained-release tip or vaccine comprises a first influenza strain, and vaccination of a subject with a dose of the first influenza strain (e.g., a first influenza A, B, C, and / or D strain as described herein) results in broad-spectrum immunity to a second influenza strain that is not present in the implantable sustained-release tip or vaccine (e.g., a drifted influenza A, B, C, and / or D virus strain as described herein).

[0188] In some embodiments, the subject (e.g., a human subject) is a pediatric subject, an adult subject, or an elderly subject. The subject may have been exposed to, infected with, and / or at risk of infection with influenza virus (e.g., a particular strain of influenza virus). This risk may be due to the subject's health or age and / or travel to an area where a particular influenza virus strain is prevalent.

[0189] In some embodiments, the present invention provides a method for controlled or sustained release of a vaccine in a subject. Compared with the traditional burst release administration of a vaccine, the controlled or sustained release of a vaccine can achieve improved immunogenicity and / or broad-spectrum immunity. Without wishing to be bound by theory, the method and / or controlled or sustained release rate of vaccination described herein, for example, by a composition and / or microneedle as described herein, simulates the natural exposure pattern of a subject (e.g., a human subject) to a virus, and can provide enhanced immunity and / or broad-spectrum immunity to the subject, compared with the traditional single-dose vaccination method.

[0190] In some embodiments, a desired amount of at least one vaccine, antigen and / or immunogen (e.g., flu vaccine and / or coronavirus vaccine, e.g., mRNA-based vaccine) can be released from a microneedle (e.g., implantable microneedle tip) described herein in a sustained manner over a predetermined period of time. In some embodiments, at least about 5% of the vaccine, antigen and / or immunogen (e.g., flu vaccine and / or coronavirus vaccine, e.g., mRNA-based vaccine), for example, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 97%, about 98% or about 99%, or 100% of the vaccine, antigen and / or immunogen (e.g., flu vaccine and / or coronavirus vaccine, e.g., mRNA-based vaccine) can be released from a microneedle (e.g., implantable microneedle tip) over a predetermined period of time. In such embodiments, a desired amount (e.g., a dose, such as a standard dose of a vaccine) of a vaccine, antigen, and / or immunogen (e.g., a flu vaccine and / or a coronavirus vaccine, such as an mRNA-based vaccine) can be released from the microneedle within seconds, minutes, hours, months, and / or years. In some embodiments, a desired amount (e.g., a dose, such as a standard dose of a vaccine) of a vaccine, antigen, and / or immunogen (e.g., a flu vaccine and / or a coronavirus vaccine, such as an mRNA-based vaccine) can be released from the microneedle within, for example, within 5 seconds, within 10 seconds, within 30 seconds, within 1 minute, within 2 minutes, within 3 minutes, within 4 minutes, within 5 minutes, or longer after insertion into the biological barrier. In some embodiments, a desired amount (e.g., a dose, such as a standard dose of a vaccine) of a vaccine, antigen and / or immunogen (e.g., a flu vaccine and / or a coronavirus vaccine, such as an mRNA-based vaccine) can be released from the microneedles for at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 6 hours, at least about 12 hours, at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 1 week, at least about 2 weeks, at least about 1 month, at least about 2 months, at least about 3 months, at least about 6 months, or longer. In some embodiments, a desired amount (e.g., a dose, such as a standard dose of a vaccine) of a vaccine, antigen and / or immunogen (e.g., a flu vaccine and / or a coronavirus vaccine, such as an mRNA-based vaccine) can be released from the microneedles for about 1 year or more.

[0191] In some embodiments, the present invention provides methods for enhancing the immune response of a subject to a virus. In some embodiments, the presence of a cell-mediated immune response can be determined by any art-recognized method, for example, proliferation assays (CD4+T cells), CTL (cytotoxic T lymphocyte) assays (see Burke, supra; Tigges, supra), or immunohistochemistry with a tissue section of the subject to determine the presence of activated cells such as monocytes and macrophages after inoculation of the immunogen. Those skilled in the art can easily determine the presence of humoral-mediated immune responses in a subject by any mature method. For example, the antibody levels produced in biological samples such as blood can be measured by Western blotting, ELISA or other known antibody detection methods. In some embodiments, elevated hemagglutination inhibition (HAI) antibody titers can be detected in the blood of the subject throughout the flu season after immunization.

[0192] In some embodiments, the immune response and / or broad immunity is a cellular and / or humoral immune response comprising: (i) elevated hemagglutination inhibition (HAI) antibody titers detectable in the blood of the subject, e.g., detectable for at least 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, 28, 29 and / or 30 weeks or longer after immunization; (ii) elevated anti-influenza IgG titers detectable in the blood of the subject, e.g., Detectable at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and / or 12 months or longer after immunization; and / or (iii) detectable levels of antibody secreting plasma cells (ASCs) against a virus, such as influenza virus, in the subject's bone marrow, e.g., detectable at least 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, 28, 29, 30, 31, 32, 33 and / or 34 weeks or longer after immunization. In some embodiments, the elevated HAI antibody titers are against drifting influenza A, B, C and / or D strains. In some embodiments, the elevated anti-influenza IgG titers are against drifting influenza A, B, C and / or D strains. In some embodiments, the immune response is a cellular immune response, including expression of IFN in the blood of the subject. Y The levels of secretory cells are increased, for example, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and / or 12 weeks or more after immunization, for example, with a microneedle as described herein.

[0193] In some embodiments, compared to a single dose or bolus administration of the vaccine, elevated HAI antibody titers, elevated anti-influenza IgG titers, levels of antibody-secreting plasma cells (ASCs) against the virus, and / or IFN Y The levels of secreting cells are higher (e.g., 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, or 15-fold or more).

[0194] In some embodiments, broad-spectrum immunity can be characterized by measuring the seroconversion percentage of the subject. For example, broad-spectrum immunity may include a seroconversion percentage, for example, based on the elevated HAI antibody titer detectable in the subject's blood, for example, greater than about 20% (e.g., 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more than 95%, for example, 100%) 6 months after immunization. Compared with the seroconversion levels obtained by traditional burst release administration of vaccines, for example, single doses or bolus administration of vaccines, the seroconversion levels associated with broad-spectrum immunity given by using the methods, dosage regimens, microneedles and microneedle devices described herein can be higher (e.g., 1 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times or 15 times or more).

[0195] Combination therapy

[0196] Microneedles and microneedle devices (such as microneedle patches) described herein can be manufactured by accurately filling each individual microneedle tip to achieve different modes of vaccine delivery, dosage regimens, and vaccine co-administration with additional therapeutic agents. The methods of immunization, vaccine delivery, and administration described herein may include vaccine co-administration with additional therapeutic agents. In some embodiments, additional therapeutic agents may be prepared in the same tip as the vaccine. In some embodiments, additional therapeutic agents may be prepared together with the vaccine. For example, adjuvants that enhance the immune response to co-delivered antigens may be delivered in the same microneedle tip and / or vaccine. Without wishing to be bound by theory, this combination therapy may include adjuvants to drive stronger cellular immune responses and / or mucosal responses. In addition, additional influenza antigens may be delivered for heterologous "primary / boost-like" immunization, for example, primary immunization with HA antigens from various influenza strains, and booster immunization (for example, provided by controlled release or sustained release or a kinetic pattern different from "primary") with different antigens (for example, drift strains, hemagglutinin stems, m2e proteins, or NA).

[0197] Formulation compatibility may limit whether two given therapeutic agents can be co-formulated for dispensing to the same needle tip. In cases where co-formulation is not possible, the manufacturing process can be adjusted so that a first formulation is dispensed into one portion of the needle array and a second formulation is dispensed into a different portion of the needle array. Different formulations can also receive different processing after filling. For example, if the first formulation is for controlled or sustained release and the filaments will be made more insoluble by water annealing, and the second formulation is for burst release without annealing, the second formulation can be dispensed after the annealing step. The manufacturing method is flexible, so other process sequences are possible.

[0198] In some embodiments, the present invention also provides methods for combined therapy, wherein the microneedles or microneedle devices of the present invention can be manufactured to administer at least one additional therapeutic agent. Various forms of therapeutic agents can be used that can be released from the microneedles described herein into adjacent tissues or fluids when administered to a subject. In some embodiments, additional therapeutic agents can be included in the base layer and / or implantable tip.

[0199] Examples of additional therapeutic agents that can be used according to the methods of the present invention, for example, examples of additional therapeutic agents that can be incorporated into the microneedles of the present invention during manufacturing, include steroids and steroid esters (e.g., estrogen, progesterone, testosterone, androsterone, cholesterol, norethindrone, digoxigenin, cholic acid, deoxycholic acid, and chenodeoxycholic acid), boron-containing compounds (e.g., carboranes), chemotherapeutic nucleotides, drugs (e.g., antibiotics, antivirals, antifungals), enediynes (such as calicheamicin, esperamicin, danemycin, neocarzinostatin chromophores, and kedarcidin chromophores), heavy metal complexes, drugs (e.g., cisplatin), hormone antagonists (e.g., tamoxifen), nonspecific (non-antibody) proteins (e.g., sugar oligomers), oligonucleotides (e.g., mRNA sequences or antisense oligonucleotides that bind to target nucleic acid sequences), peptides, proteins, antibodies, photodynamic agents (e.g., rhodamine 123), radionuclides (e.g., I-131, Re-186, Re-188, Y-90, Bi-212, At-211, Sr-89, Ho-166, Sm-153, Cu-67, and Cu-64), toxins (e.g., ricin), and transcription-based drugs.

[0200] Example Kit

[0201] In some embodiments, the present invention relates to a package or kit comprising a microneedle as described herein (e.g., a microneedle comprising a vaccine, antigen and / or immunogen as described herein, such as an influenza virus). In some embodiments, the present invention relates to a package or kit comprising a vaccine as described herein (e.g., a vaccine, antigen and / or immunogen as described herein, such as an influenza virus). In some embodiments, the kit may further include an additional therapeutic agent for combined treatment with the microneedle. In some embodiments, the kit may further include a disinfectant (e.g., an alcohol swab). In some embodiments, such packages and kits described herein may be used for vaccination purposes, for example, to achieve broad-spectrum immunity in a subject as described herein.

Claims

1. A patch and applicator system comprising: A drug patch, the drug patch comprising a sheet of flexible material; as well as An applicator, which is used to hold the drug patch and apply the drug patch to the patient's skin, comprising: an outer body portion having a substantially cylindrical shape and a hollow interior; a piston portion slidably coupled to the hollow interior of the outer body portion; a compressible member disposed within the outer body and configured to apply downward pressure to the piston; and an actuator positioned below the piston and slidably engaged with the hollow interior and extending from the bottom of the outer body such that upward pressure on the actuator pushes the piston upward and compresses the compressible member; wherein the piston and the hollow interior of the outer body portion are slidably connected with a protrusion and a cam path, and wherein the cam path forms a continuous loop such that upward pressure on the piston portion causes the piston to compress the compressible member, moving the piston upward into the hollow interior of the outer body portion until a protrusion reaches the top of the cam path and engages a downward portion of the cam path, thereby releasing the piston into the downward portion of the continuous loop to release the piston and force the piston downward; and The drug patch is held in an annular holder at the bottom of the actuator and is pushed downward through the annular holder and onto an object that is in contact with the bottom surface of the annular holder.

2. The system according to claim 1, wherein: The compressible member includes a spring.

3. The system according to claim 1 or 2, wherein: The annular retainer is removable from the actuator.

4. The system according to claim 1 or 2, wherein: The annular retainer is integrally connected to the actuator.

5. The system according to any one of claims 1 to 4, wherein: The piston and the hollow interior of the outer body portion are slidably connected with at least one additional protrusion and at least one additional cam path.

6. The system according to any one of claims 1 to 4, wherein: The protrusion and the cam path include a protrusion extending from a side wall of the piston and a cam path formed along an inner wall of the outer body portion.

7. The system according to claim 5, wherein: Each protrusion and cam path includes a protrusion extending from a side wall of the piston and a cam path formed along an inner wall of the outer body portion.

8. The system according to any one of claims 1 to 7, wherein: The continuous loop of the cam path comprises a substantially parallelogram-shaped loop.

9. The system according to any one of claims 1 to 8, wherein: The actuator is slidably engaged with the hollow interior along a connection between at least one protrusion of the actuator and at least one cam path of the hollow interior.

10. The system according to claim 9, wherein: The at least one cam path of the actuator and the at least one cam path of the hollow interior are configured to allow substantially linear sliding of the actuator relative to the hollow interior without requiring rotation of the actuator.

11. The system according to any one of claims 1 to 10, wherein: The piston is rotatably movable within the hollow interior.

12. The system according to any one of claims 1 to 11, wherein: The annular retainer has a solid edge portion and an open center portion.

13. The system according to claim 12, wherein: The solid edge is substantially circular.

14. The system according to claim 12 or 13, wherein: The drug patch and the annular retainer are sized such that the patch is larger than the open center portion in at least one dimension so that the drug patch can be positioned on top of the annular retainer without passing through the center portion.

15. The system of claim 14, wherein: The drug patch has a degree of flexibility such that releasing the downward pressure applied to the drug patch by the piston causes the drug patch to flex sufficiently to allow the drug patch to pass through the central portion of the annular retainer.

16. The system according to any one of claims 1 to 15, wherein: The drug patch is a flat sheet in the form of a circle, triangle, square, polygon or oval.

17. A system according to any one of claims 1 to 16, wherein: The drug patch has one or more notches along its periphery to facilitate bending of the patch about the periphery of the patch.

18. A system according to any one of claims 1 to 17, wherein: The patch includes a microneedle patch.

19. The system of claim 18, wherein: The patch is a fibroin-based microneedle patch.

20. The system according to claim 18 or 19, wherein: The microneedle patch includes biodegradable microneedles.

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