Microneedle 16 can therefore provide a sharp needle tip along with enhanced mechanical performance through a filleted base enables successful tissue penetration, a pyramid head that serves as the bioactive material dosage form where the bioactive material(s) are incorporated into dissolving or degrading biomaterial matrix, and an undercut stem portion that improves the mechanical performance during penetration while ensuring tissue retention during implantation, As with microneedle 14, the quickly dissolving layer along with the mechanical mismatch between dissolving and non-dissolving layers can facilitate quick separation of pyramid tips and the non-dissolvable undercut stem portion prevents back diffusion of the embedded bioactive material during both fabrication and implantation processes. In this embodiment, the backing layer can conform to non-uniform skin topography better and, if non-dissolvable, it can help prevent absorption of humidity which may result in excessive curvature of the backing layers and render MNA applications suboptimal.

FIG. 2A-2C illustrate the ability of a microneedle array 50 that has microneedles 52 and a conformable backing layer 34 to bend, and after bending return to an original state, or at least a state that is closer in shape to the original state than the bent state.

It should be understood that any combination of the features disclosed in FIG. 1—and throughout this application—is contemplated. For example, the conformable backing layer of microneedle 16 and the quickly dissolving layer 30 of microneedles 14, 16 can be used in combination with any of the other structures disclosed herein (e.g., microneedles 10, 12).

In addition, the processes disclosed herein can be used to create a microneedle that is formed entirely from non-dissolving materials for use as a coated MNAs, tissue adhesives (patches), and/or microbarbs. Non-dissolvable undercut microbarb or microneedle geometries have been created previously using additive manufacturing or mechanical micromachining processes. However, high-throughput manufacturing of those geometries is hindered due to lack of effective micromolding processes.

Micro-Additive Manufacturing of MNAs

In some embodiments, the MNAs can be formed using additive manufacturing (AM), including micro-additive manufacturing (μAM) techniques. The methods and systems described herein can allow medical researchers with little microfabrication expertise to directly produce their MNA designs from a computer-aided design (CAD) drawing without the complex requirements of the subtractive fabrication processes. The μAM techniques described herein provide an effective means for fabrication of novel MNAs designed specifically for effective cutaneous and non-cutaneous drug delivery.

In some embodiments disclosed herein, the MNA designs included uniquely-shaped micron-scale needles that comprising sharp pyramid heads and undercut stems with filleted bases to ensure successful skin penetration and retention. Unlike conventional MNAs, the MNAs disclosed herein are fabricated by a three-dimensional (3D) μAM approach with 3D direct laser writing, which offers transformative potential for the MNA field with its unparalleled level of simplicity and design capabilities.

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

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

In some embodiments, the MNAs can incorporate a model antigen Ovalbumin (OVA)±a model adjuvant Poly(I:C) from a biodissolvable material composition (70% CMC/30% Treh) using a spin-casting method. Furthermore, the proposed MNA designs, along with favorable mold materials, strategically enabled direct fabrication steps without interfering with molding processes. The fabricated MNAs are particularly effective in penetrating through the stratum corneum of living human skin to deliver their biocargos to the cutaneous microenvironments. These unique MNAs fulfill the geometric and mechanical-strength requirements for effective skin penetration for cutaneous vaccination, thereby presenting an alternative promising approach for skin-targeted immunization strategies.

Exemplary Microneedles and Arrays, and Manufacturing Systems Thereof

FIGS. 3A and 3B illustrate an exemplary microneedle array (MNA) (100) that comprises microneedles with a sharp-tipped pyramid head (102) and an undercut stem portion (104). In addition, the microneedles include a filleted base (106).

The methods and systems disclosed herein enable reproducible fabrication of high-quality, tip-loaded dissolving MNAs with undercut features from different and widely-used dissolving microneedle materials, including CMC, PVP, Silk, HA, CMC/Trehalose, CMC/Glucose, CMC/Sucrose, PVP/PVA, and any other moldable biodissolvable compositions.

In this embodiment, the microneedle has a height (110) that is between 50-1500 μm, such as 750 μm in height, and an apex angle (A) of the pyramid head that is between 10°-60, such as 30°.

In one embodiment, the stem portion of the microneedle can have a width (112) that is between 50-500 μm, such as 150 μm. The stem portion (104) can extend from the bottom region of a three-dimensional (3D) pyramid head to a backing layer (108) of the microneedle with a radius filleted connection that ranged from 15-75 μm, such as 35 μm. A width (114) of the bottom region of the pyramid head can be, for example, between 100-400 μm, such as 250 μm×250 μm base area.

In some embodiments, the tip-to-tip distance (116) between the microneedles in the array can be between 100-800 μm, such as 650 μm. In some embodiments, the MNA can include between 1-1000 microneedles, such as 25 microneedles in a 5×5 array configuration on a backing layer with an area of 4.75 mm×4.75 mm.

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

The undercut, or anchor feature, can improve skin retention during application and, using the novel methods and systems disclosed herein, can be achieved without also interfering with the processing steps, thereby allowing direct removal of the MNAs from the molds.

The three-dimensional micro-additive manufacturing (3D-μAM) approach described herein allows for the creation of unique MNA designs from a 3D-CAD drawing. Furthermore, 3D-μAM can enable medical researchers with little or no microfabrication expertise to design and manufacture MNA-based drug delivery platforms with application-oriented optimizations.

Exemplary manufacturing systems and processes are presented in a flow chart in FIG. 4A and graphically illustrated in FIG. 4B. As shown in FIGS. 4A and 4B, the process can comprise the following steps to create novel fast-dissolving MNAs while simultaneously achieving high-throughput fabrication. First, a 3D-CAD drawing (132) of the MNA design can be prepared (process step 120). Second, a direct production of the master MNA (134) from the CAD drawing can be produced by 3D direct laser writing using a non-dissolvable resin (e.g., IP-S photoresist) (process step 122). The 3D-μAM manufacturing system can be, for example, a Nanoscribe 3D printing system (136).

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

Fourth, the MNA master molds can be created with multiple master MNA replicas (e.g., six replicas) onto 3D-printed MNA holders (process step 126). The 3D printed MNA holder (142) can be formed of, for example, a resin material. The combination of a plurality of master MNA replicas together onto, for example, 3D-printed MNA holders can allow for the creation of larger MNAs and improve productivity.

Fifth, the MNA production molds (144) can be manufactured from the elastomer PDMS using micromolding (process step 128). Sixth, tip-loaded, dissolving MNAs (150) can be fabricated (process step 130). The MNAs can have undercut microneedles incorporating one or more bioactive material, such as a vaccine or any other bioactive material from a water-soluble biocompatible material (e.g., a composition of CMC and Treh) through a multiple-step spin-casting method using a centrifuge.

For example, a bioactive component (e.g., vaccine) can be spin-casted to the tip of the PDMS production molds and the dissolvable hydrogel (e.g., CMC/Trehalose) can be spin-casted into the production molds to serve as the structural material of microneedles and to form the backing layer of the MNAs. In some embodiments, the master MNA, the MNA master molds including replicas of the master MNA, and the elastomer production molds can be reused for a large number of processing cycles, thereby greatly reducing the fabrication costs for MNAs with unique designs and improving productivity.

FIGS. 5A-5E show final products corresponding to different manufacturing or processing steps. FIG. 5A shows an optical microscope image of the master MNA (134) created using 3D direct laser writing and FIG. 5D shows an optical microscope image of an individual undercut needle on the 3D printed master MNA (134). Specifically, the master MNA was fabricated from IP-S photoresist by 3D direct laser writing. IP-S is a specific material designed for 3D laser lithography and provides high resolution and mechanical integrity for micro-and nano-structures. 3D laser lithography based on two-photon polymerization provided an effective means for fabrication of undercut MNA designs with smooth edges and sharp tips, and without any unwanted residues (e.g., machining chips).

FIG. 5B shows an optical microscope image of the replica (140) of the master MNA created through a two-stage micromolding strategy: elastomer molding combined with UV-curable micromolding and FIG. 5E shows an optical microscope image of an individual undercut needle on the replica (140) of the master MNA. The master MNA was produced using a two-step micromolding process. The IP-S master MNA was used to fabricate PDMS flexible mold through elastomer molding and then the PDMS mold was used to manufacture several Vero White MNA replicas through UV-curable micromolding. VeroWhite resin is a wear-resistant, acrylic-based photo-polymer that can be used with 3D Polyjet printers.

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

As such, these processing steps, along with high geometric capability of 3D direct laser writing, resulted in a scalable and effective MNA manufacturing strategy. Furthermore, rapid replication of the 3D printed master MNA using a wear-resistant moldable material improved productivity substantially. Other undercut features using different microneedle and elastomer mold materials, consistent with the processes and systems described herein, are also possible.

Upon fabrication of the MNA master molds with six MNA replicas, dissolving MNAs that integrate the vaccine in the tip portion of the needles were fabricated using the conventional three-stage manufacturing strategy through master mold to production mold to final dissolvable MNAs.

As discussed in more detail below, dissolving MNAs that incorporated the vaccine (10 μg OVA±25 μg Poly(I:C)) in the tip portion of the undercut needles were fabricated through the spin-casting process from two different material compositions (i.e., CMC/Trehalose and PVP/PVA). Furthermore, tip-loaded CMC/Trehalose MNAs with undercut microneedles integrating a colored model drug (e.g., Doxorubicin) were fabricated to facilitate imaging and demonstrate compatibility with chemotherapeutic agents. The systems and methods described herein enabled effective and rapid fabrication of tip-loaded dissolving MNAs with undercut microneedles from different dissolvable material compositions.

FIG. 6A shows an optical microscope image of final dissolving (CMC/Treh) MNAs incorporating biocargos (e.g., OVA+Poly(I:C)). FIG. 4B shows an optical microscope image of dissolving, tip-loaded PVP/PVA MNAs with undercut microneedles integrating OVA, and FIG. 4C shows an optical microscope image of an individual tip-loaded CMC/Treh undercut microneedle incorporating Doxorubicin as a colored drug to facilitate imaging.

EXAMPLE 1
Manufacturing of Exemplary MNAs

An exemplary process for creating an MNA is described herein.

Fabrication of master MNA. A unique MNA design was directly created from the 3D-CAD drawing shown in FIG. 1 using 3D laser printing (Nanoscribe Photonic Professional, GT) with the photopolymetric resist IP-S. The Nanoscribe printing system was equipped with a laser generator, an optical cabinet, a Zeiss optical microscope attached to a lens to focus the laser beam, a Galvo mirror system to direct the laser-beam scanning, a piezoelectric stage for precise motion control, and an operation software (Nanowrite) to execute 3D printing. The whole system was placed on an optical table to eliminate vibrations during the printing process.

To fabricate the master MNA, the MNA design was converted into ‘STL’ (StereoLithography) format. Subsequently, the STL file was loaded into the specialized software (DeScribe, Germany) of the Nanoscribe system to select the processing conditions (i.e., the distance of slicing, hatching, and splitting). Finally, the STL file was converted into ‘GWL’ (General Writing Lithography) format to be exported in the Nanowrite software for printing the master MNA. The master MNA was fabricated using Galvo-scan mode in XY plane and piezo-scan mode in Z direction. The master MNA was split into blocks of 220 μm×220 μm×200 μm within the working range and then stitched together. The laser power and writing speed was set to be 100 mW and 6 cm/s, respectively. The minimum and maximum slicing distances of 0.3 μm and 0.5 μm, respectively, were used. The master MNA was then printed through two-photon polymerization of the IP-S photoresist by a femtosecond pulsed laser at a wavelength of 750 nm using a unique deep-in-liquid mode with the objective of 25× and NA0.8 in Shell and Scaffold mode.

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

Replication of master MNA. To replicate the master MNA with high-fidelity using a UV-curable resin, a two-stage micromolding method was performed. First, an elastomer mold which is negative of the master MNA was manufactured from polydimethylsiloxane (PDMS) using a micromolding approach. Elastomer molding using PDMS provides an accurate and reproducible replication of high-fidelity micron-scale structures. Briefly, the master MNA was mounted in a petri-dish with a diameter of 5 cm and PDMS was obtained using the two-component curable silicone elastomer, SYLGARD® 184 (Dow Corning), by mixing the base material with a curing agent in 10:1 SYLGARD®-to-curing agent ratio. Subsequently, the mixture was poured over the master MNA mounted into the petri-dish and degassed for 15 min. Next, the master MNA with the degassed mixture was placed in an oven and cured at 70° C. for 1 h. The cured PDMS was cooled down to room temperature for 5 min and then separated from the master MNA to obtain the negative PDMS mold.

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

Creation of MNA master molds. To facilitate scalable manufacturing of dissolving MNAs, the MNA master molds were created through assembling six replicas of the master MNA onto the MNA holders fabricated by Stratasys® from a non-dissolvable photo-polymer (VeroWhite) using a high-resolution (16 _82 m) Polyjet 3D printing system (Objet Connex 500 multi-material). The 3D model of the MNA holders created using SolidWorks 2018 CAD software and then converted into the ‘STL’ (StereoLithography) file format. Subsequently, the specialized software (Objet Studio) sliced this 3D model into 2D cross-sectional layers, creating a computer file that was sent to the 3D printer system. The channels in the 3D printed MNA holder were designed and fabricated to serve as raised pockets in the MNA production molds to assist as reservoirs for both the bioactive cargo (e.g., vaccine) and the structural hydrogel material of dissolving MNAs during the spin-casting process. The created MNA master molds were baked at 80° C. overnight in a vacuum oven to facilitate effective fabrication of elastomer MNA production molds.

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

Production of dissolving MNAs. To fabricate tip-loaded, dissolving MNAs with undercut microneedles incorporating OVA±Poly(I:C), a biodissolvable material composition that included carboxymethylcellulose (CMC, cat #C5678, Sigma-Aldrich, St Louis, MO) and trehalose (Treh, Cat #T9531, Sigma-Aldrich) was used. To prepare the hydrogel form of the structural material of dissolving MNAs, the CMC and Treh powders were thoroughly mixed based on the weight ratio of 70% and 30%, respectively. This powder mixture was then added to endotoxin-free water (HyClone HyPure Cell Culture Grade Water) and thoroughly mixed to achieve a 30% w/w solute concentration. The prepared hydrogel was refrigerated at 4° C. for 24 h for the mixture to equilibrate. Tip-loaded CMC/Treh-MNAs with the unique designs were then manufactured through a multiple-step spin-casting technique using a centrifuge (Thermo Fisher Scientific Sorvall Legend XTR with Swinging Bucket Rotor TX-750).

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

For MNAs integrating OVA+Poly(I:C), after loading OVA, 5 μL of Poly(I:C) solution (62.5 mg/mL Poly(I:C) in endotoxin-free water) was dispensed over each of the MNA on the PDMS production molds, and the production molds were centrifuged for 1 min at 20° C. and at 4500 rpm to fill the microneedle-shaped cavities. Once the production molds were filled, the excess Poly(I:C) solution within the reservoir was recovered. The production molds were again centrifuged for 30 min at 20° C. and at 4500 rpm to ensure that Poly(I:C) cargo was also located at the pyramid portion of the needle-shaped cavities in the production molds. After locating the biocargos at the tip of microneedles, 40 μL of hydrogel (30% w/w 70:30 CMC:Treh) was loaded over each of the MNAs on the PDMS production molds to fill the microneedle-shaped geometries in the production molds and to form the backing layers of the MNAs. The hydrogel-loaded production molds were then centrifuged for 5 h at 20° C. and at 4500 rpm to obtain dissolving MNAs loaded with 10 μg OVA±25 μg Poly(I:C). Due to the specific geometry of the MNA production molds, each replication produced six arrays of OVA±Poly(I:C)-loaded dissolving MNAs simultaneously.

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

Cutaneous Delivery Using Exemplary MNAs

Preparation of ex vivo human skin explants. Human skin explants were prepared from deidentified healthy donors undergoing plastic surgery acquired through the Pitt Biospecimen Core and used according to University of Pittsburgh Medical Center guidelines. Tissue was rinsed in 70% ethanol and then in phosphate-buffered saline (PBS). Human skin explants (approximately 1 mm thick) were harvested using a Silver's miniature skin graft knife (Padgett, Integra Miltex, Plainsboro, NJ), and then cut into 20 mm×20 mm square pieces. The resulting human skin samples were comprised of unaltered epidermis and a thin layer of underlying dermis, and maintained as explants in normal physiological state by culture at an air-fluid interface.

Imaging analysis. To evaluate MNA-directed intradermal bioactive materials (e.g., vaccine) delivery to living human skin explants, a number of imaging analyses was performed. Tip-loaded CMC/Treh-MNAs integrating a colored cargo (i.e., Allura Red R40 dye) were fabricated using the manufacturing strategy described above. Prior to application of MNAs to the human skin explants, MNAs were imaged using bright-field optical microscopy.

Subsequently, MNAs were applied to human skin explants and removed after 10 min. The targeted human skin regions were then examined under a bright-field microscope to image the patterns of the colored biocargo deposited from CMC/Treh-MNAs into the human skin. Remaining MNA materials were also imaged using optical microscopy after human skin applications. For further qualitative assessment of MNA-directed intradermal vaccine delivery to human skin, CMC/Treh MNAs that incorporate both Alexa555-labeled OVA and Alexa488-labeled Poly (I: C) were fabricated, applied to human skin for 10 min, and removed. The targeted human skin explants were then histologically analyzed. Briefly, the MNA-treated human skin samples were fixed in 2% paraformaldehyde followed by immersion in sucrose solution, with 3 changes of this solution over 24 h. Tissue sections were then flash frozen in optimum cutting temperature (OCT) histology compound, and cryo-sectioned into approximately 10 μm thick sections. The sectioned human skin samples were counter-stained using nuclear DAPI fluorescent dye. The stained sections were then imaged using a Nikon transmission fluorescent microscope to detect Alexa555-OVA and Alexa488-Poly(I:C) into the cross-sections of human skin, as well as using bright field microscope to better demonstrate the stratum corneum breaching.

MNA-Directed Skin Immunization in Vivo

Mice and animal husbandry. Female C57BL/6J mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and used at 8-10 weeks of age. Mice were maintained under specific pathogen-free conditions at the University of Pittsburgh, and all experiments were conducted in accordance with the institutional animal care and use committee (IACUC) guidelines.

In vivo IVIS imaging. In vivo intradermal vaccine delivery using novel dissolving MNAs was demonstrated using a C57BL/6J mouse. Tip-loaded CMC/Treh-MNAs integrating both Alexa555-labeled OVA and Alexa488-labeled Poly(I:C) were created using the manufacturing strategy described above and applied to the abdomen of the mouse that was temporarily anaesthetized via isoflurane inhalation for 10 min. Next, MNAs were removed and the mouse was returned to their normal activity. OVA+Poly(I:C) loaded MNAs were imaged before and after in vivo application. Imaging of the MNA-treated mouse was performed on the IVIS 200 in vivo imaging system (PerkinElmer) using the filters to assay for fluorescent-labeled Poly(I:C) and OVA the MNA application site. The image was post-processed using Living Image software (PerkinElmer).

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

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

To assess OVA-specific cytotoxic T-cell (CTL) activity, splenocytes from naïve mice were pulsed with 2 μg/ml OVA257-264 (SIINFEKL) peptide, or left unpulsed for 1 h. Antigen pulsed splenocytes were washed and stained with high concentration carboxyfluorescein succinimidyl ester (CFSE, 10 μM), while unpulsed splenocytes were labeled with low concentration CFSE (1 μM) for 15 min at 37° C. A 1:1 mixture of pulsed target cells and unpulsed control cells (107 each) was intravenously (IV) injected into immunized and naïve mice. Twenty hours after adoptive transfer, spleens of mice were isolated, and killing of target cells was evaluated by comparison of the antigen pulsed and unpulsed populations by flow cytometry to quantify OVA-specific killing of the high CFSE labeled SIINFEKL-pulsed targets. Specific lysis was calculated and expressed as a percentage of maximum lysis as % Lysis={1−[(mean CFSElow/CFSEhigh ratio from naïve mice)/(CFSElow/CFSEhigh ratio from vaccinated mouse)]}×100.

Intradermal and Non-Cutaneous Uses

In addition to cutaneous vaccination, the MNAs described herein can be used for a broad range of intradermal and non-cutaneous (e.g., ocular and cardiac tissues) drug delivery applications. As shown in