Design and fabrication of dissolving polymer microneedles
The polymer material, microneedle geometry and device fabrication process were designed to safely encapsulate influenza virus while preserving its antigenicity, insert into skin without mechanical failure, and rapidly dissolve in skin, leaving behind safe dissolution products. The resulting microneedles measured 650 μm tall with sharp tips tapering to a 10 μm radius of curvature () and were assembled into an array of 100 needles () that encapsulated 3 μg of inactivated influenza virus vaccine per patch.
Dissolving polymer microneedle patches
These microneedles were fabricated by room temperature photopolymerization of a liquid monomer (vinyl pyrrolidone) within a microneedle mold to form polyvinylpyrrolidone (PVP) microneedles that encapsulate the lyophilized vaccine. PVP was chosen as the structural material for the polymer microneedles used in this study, because it is biocompatible, mechanically strong and highly water soluble20
. Moreover, PVP polymer microneedles were fabricated by a gentle, room-temperature photopolymerization process, which avoids need for organic solvents or elevated temperatures that can damage vaccine or other biomolecule stability.
Insertion and dissolution of microneedles in skin
The resulting microneedles were able to be inserted into skin with gentle force applied by thumb (). We determined the fracture force of microneedles to be 0.13±0.03 N per needle, which provides a two-fold margin of safety over the force (0.058 N per needle) required for insertion into skin using microneedles of this geometry, according to previous measurements21
. Upon insertion into porcine cadaver skin, microneedles penetrated to a depth of approximately 200 μm and deposited their encapsulated payload within epidermis and upper dermis (). This localization is likely to be similar in human skin, which has comparable thickness to porcine skin22
Delivery to skin using microneedles
To characterize kinetics of dissolution in skin, microneedles were inserted into porcine skin and monitored over time. Significant dissolution occurred within 1 min, and after 5 min the microneedles were 89±3% (by mass) dissolved (). Given the similarity of porcine and human skin, we expect that microneedle dissolution in human skin could also be complete within just a few minutes. Because vaccination experiments in this study used mouse skin, we also measured dissolution kinetics of dissolving microneedles encapsulating the viral antigen in mice. In this scenario, microneedle dissolution was slower, but nonetheless increased with time (P<0.05), depositing 34±17%, 63±10% and 83±6% in the skin after 5, 10 and 15 min, respectively, and leaving almost no residue on the skin surface ().
To assess stability of inactivated influenza vaccine in dissolving microneedles, we identified two steps during fabrication of PVP microneedles that might cause damage: initial lyophilization of vaccine and subsequent encapsulation within microneedles during polymerization.
To isolate effects of lyophilization and PVP, inactivated influenza virus was administered IM in mice (i) as the original vaccine solution, (ii) after lyophilization, (iii) as the original vaccine solution mixed with PVP, and (iv) after lyophilization and encapsulation within PVP microneedles. Compared to naïve animals, all four vaccinated groups showed elevated influenza-specific IgG titers and hemagglutination inhibition (HAI) titers (, P<0.01). Among the four vaccinated groups, there was no significant effect of vaccine processing or formulation on IgG or HAI titers (P>0.05).
Humoral immune responses
The efficacy of skin immunization with dissolving microneedles was determined in BALB/c mice that received a single dose of 6 μg of whole encapsulated inactivated influenza virus. The microneedle patches were applied on the caudal dorsal area of skin for approximately 15 min, which was sufficient to dissolve the microneedles and deliver at least 80% of the antigen into skin. Induction of humoral immune responses using dissolving microneedles was compared at the same dose to those observed by IM immunization, which is the standard influenza vaccination method (). Blood was collected on days 14 and 28 post-immunization to determine levels of anti-influenza-specific antibodies. Mice immunized with microneedles demonstrated a significant increase of anti-influenza IgG titers by day 14 (, P<0.0003). Titers were at similar levels for both IM and microneedle groups at day 28 (p=0.9).
Microneedle immunization studies
We also determined levels of influenza-specific isotypes, IgG1 and IgG2a, at 14 and 28 days after immunization. At day 14, IM-immunized mice showed significantly higher IgG2a responses than the microneedle group (p=0.0006), whereas the microneedle group had more pronounced IgG1 titers than the IM group (p=0.03). At day 28 there were no significant differences in the isotype levels between the groups. Thus, the IM group had Th1 biased responses early after immunization (IgG1/IgG2a=0.2) but isotype levels were similar one month later (IgG1/IgG2a=0.9). In contrast, the microneedle group showed a slight predominance of IgG1 production over time (IgG1/IgG2a in the range of 1.35–1.53) (,).
Long-lived immune responses
HAI activity is generally used as the serological measure for functional antibodies associated with protection. We observed high HAI titers after one immunization (). HAI titers detected in the microneedle group were similar for the two time point bleedings and to IM group titers, demonstrating that a single microneedle immunization induced high levels of functional antibodies.
Protection against lethal viral challenge
To determine whether microneedle immunization can confer protective immunity, the immunized groups were challenged with 5xLD50 of mouse-adapted PR8 influenza virus 30 days after vaccination. All immunized animals survived challenge () and lost <5% body weight (), showing that vaccine delivery with dissolving microneedles provided protection equal to the IM group. In contrast, the unimmunized group did not survive beyond 6 days post-challenge.
We then investigated the ability of challenged mice to clear influenza virus from the lung 90 days after vaccination to assess longevity and efficiency of recall responses. IM immunized mice showed a 103 decrease in lung viral titers compared to unimmunized infected mice, whereas microneedle-immunized mice showed a dramatic 106 decrease in lung viral titers (). Because challenge of animals took place three months after vaccination, we observed that microneedle immunization induced more robust recall responses than IM vaccination as shown by faster virus clearance.
Recall immune responses
To evaluate induction of local immune responses, we measured influenza-specific IgG and IgA titers in lungs of challenged mice 90 days post-immunization. We found that sIgA levels were modestly increased in vaccinated groups and were similar among microneedle and IM groups (). Lung IgG titers were also similar in microneedle and IM immunized mice, including IgG1 and IgG2a isotype profiles (). Systemically, we observed that challenged mice had serum influenza-specific IgG titers similar to those observed 28 days after immunization, with no significant differences among immunized groups (). Serum HAI titers also reached similar levels in all immunized challenged groups, consistent with total antibody levels (). Although we noted an increase of IgG1 titers post-infection in vaccinated animals, microneedle-immunized mice had a higher IgG1/IgG2a ratio than the IM group as observed in pre-challenge samples (). Thus, changes in antibody levels were consistent with protective responses in immunized mice. Overall, these data demonstrate that microneedle vaccination induced similar antibody recall responses compared to IM vaccination.
Antibody-secreting cells (ASC) are partly responsible for recall immune responses that confer protection against influenza infection. Mice challenged 90 days after immunization were examined for influenza IgG ASC in spleen and lungs on day 4 post-infection. In spleen, ASC numbers were elevated in both the microneedle and IM groups; despite lack of noticeable differences between groups, the microneedle group was the only one showing significantly higher numbers of ASC than naïve or infected mice (, P<0.03). In lungs, we observed that the microneedle and IM groups had 3–5 times higher ASC numbers than unimmunized infected or naïve mice. These results suggest that a skin vaccination route using dissolving microneedles induced sustained humoral immune responses in lungs at least as strong as responses induced by IM immunization ().
Induction of systemic cytokine responses
We next investigated induction of cellular immune responses systemically upon challenge 90 days post-immunization. We re-stimulated splenocytes isolated from challenged mice on day 4 with HA Class I (HA I) and II (HA II) restricted peptides or inactivated influenza virus for 48 h and 72 h to determine the contribution of CD4+
T lymphocytes secreting interleukin-4 (IL-4) and interferon-γ (IFN-γ) (). IL-4 secretion was higher in the IM group in the presence of Class I or Class II peptides, although increases were more prominent with Class I, suggesting increased CD8+
T cell-derived response (). In contrast, IFN-γ levels secreted by CD8+
cells were 2 to 3-fold higher in the microneedle group when compared to IM (). Naïve mice did not show any differences in cytokine levels from unimmunized infected mice (data not shown). Elevated IFN-γ levels in microneedle-immunized mice suggest that microneedle immunization generates a stronger T cell helper type 1 and effector response, which are necessary to promote antibody production and support cytotoxic activity, events that are crucial for viral clearance23
Cellular immune responses after challenge
Assessment of cellular immune responses in lungs
To assess cellular immune responses elicited in the mucosal compartment, we re-stimulated lung cell suspensions in vitro
with inactivated A/PR/8/34 virus and assessed levels of interleukin-21 (IL-21), IFN-γ, tumor necrosis factor-α (TNF-α), and interleukin-12p70 (IL-12p70). IL-21 is a pleiotropic cytokine known to upregulate genes associated with innate immunity and Th1 responses24
as well as regulating B cell isotype class switching25
. It also augments IFN-γ production in vitro
when combined with other cytokines26
. We found that IL-21 level in lungs of IM-vaccinated mice was significantly higher than other groups (, P
=0.0211), with IFN-γ production correspondingly upregulated in the same group (). Unimmunized infected mice showed highest IFN-γ and TNF-α levels (), consistent with stronger inflammatory reaction in animals not protected by vaccination. Interestingly, both IM (P<
0.0001) and microneedle (P<
0.0005) groups had significantly higher IL-12p70 production than naïve or infected groups, which correlates with the high INF-γ which was more prominent in the IM group ().
Levels of IFN-γ, IL-12p70 and IL-21 induced after polyclonal re-stimulation in lung were higher in the IM compared to microneedle group, which suggests stronger local Th1 response in the MN group upon challenge. In contrast, influenza virus MHC Class I and II restricted T cell responses were increased in spleen of microneedle-immunized groups, indicative of increased recall CD4+ and CD8+ T cell responses systemically. Increased IFN-γ production in the microneedle-immunized group may reflect enhanced generation and maintenance of memory T cells that are responsible for increased virus clearance observed in lungs when compared to the IM group. Overall, these data demonstrate that microneedle immunization can generate a robust cellular and humoral immune response similar to that observed with the conventional IM route, and suggest that microneedle immunization can establish a sustained and broader immune response.
Comparison of dissolving polymer microneedles and coated metal microneedles
As a final set of experiments, we compared dissolving polymer microneedles used in this study to coated metal microneedles used previously13–15
by vaccinating mice using each of these microneedle technologies and measuring humoral and cellular immune responses after two weeks (see Supplementary Study
). Humoral immune responses were similar (Supplementary Fig. S1
), but cellular responses differed (Supplementary Figs. S2 and S3
), most notably shown through increased IL-4 and IFN-γ production from inguinal lymph node cells in response to inactivated influenza virus stimulation in mice vaccinated using dissolving polymer microneedles compared to coated metal microneedles. This result suggests that dissolving microneedles not only offer advantages over IM injection, but also represent an improvement over coated metal microneedles.