Nanoporous Microneedle Arrays Effectively Induce Antibody Responses against Diphtheria and Tetanus Toxoid

Abstract

The skin is immunologically very potent because of the high number of antigen-presenting cells in the dermis and epidermis, and is therefore considered to be very suitable for vaccination. However, the skin's physical barrier, the stratum corneum, prevents foreign substances, including vaccines, from entering the skin. Microneedles, which are needle-like structures with dimensions in the micrometer range, form a relatively new approach to circumvent the stratum corneum, allowing for minimally invasive and pain-free vaccination. In this study, we tested ceramic nanoporous microneedle arrays (npMNAs), representing a novel microneedle-based drug delivery technology, for their ability to deliver the subunit vaccines diphtheria toxoid (DT) and tetanus toxoid (TT) intradermally. First, the piercing ability of the ceramic (alumina) npMNAs, which contained over 100 microneedles per array, a length of 475 µm, and an average pore size of 80 nm, was evaluated in mouse skin. Then, the hydrodynamic diameters of DT and TT and the loading of DT, TT, and imiquimod into, and subsequent release from the npMNAs were assessed in vitro. It was shown that DT and TT were successfully loaded into the tips of the ceramic nanoporous microneedles, and by using near-infrared fluorescently labeled antigens, we found that DT and TT were released following piercing of the antigen-loaded npMNAs into ex vivo murine skin. Finally, the application of DT- and TT-loaded npMNAs onto mouse skin in vivo led to the induction of antigen-specific antibodies, with titers similar to those obtained upon subcutaneous immunization with a similar dose. In conclusion, we show for the first time, the potential of npMNAs for intradermal (ID) immunization with subunit vaccines, which opens possibilities for future ID vaccination designs.

Keywords: antigen release; diphtheria; humoral immune response; intradermal vaccination; nanoporous microneedles; tetanus.

Figure 1

(A,B) Brightfield microscopy images of a nanoporous microneedle array (npMNA) from the needle-side with microneedles with a length of 475 µm and a density of 105 microneedles/array. (C) Microneedle applicator design that was used to apply npMNAs onto mouse ears. Upon lowering the applicator lid, a microneedle array is pierced into the skin by impact application, and the npMNA is subsequently held onto the skin by force (4 N). (D) Experimental setup for immunization studies. Immunization with either npMNA or hydrodermic needle on days 0, 21, and 42. Blood samples for serum were collected on days −1, 20, 41, and 47, at which the experiment was terminated and subsequently, spleens were collected.

Figure 2

(A) Bruker analysis was used for geometry and surface analysis and to measure the distance between the microneedle backplate and microneedle tip. The color is indicative for the size of the substrate-fillable microneedles. (B) Brightfield microscopy images of a nanoporous microneedle array (npMNA) of which only the microneedle tips are loaded with a trypan blue solution. (C) Representative image of a trypan blue piercing assay of ex vivo murine ears with an npMNA using the uPRAX applicator.

Figure 3

(A) Hydrodynamic diameter of diphtheria toxoid (DT) (8.72 ± 2.83 nm, mean ± SD, n = 3). (B) Hydrodynamic diameter of tetanus toxoid (TT) (13.5 ± 5.6 nm, mean ± SD, n = 3). (C) Release of DT and TT in release buffer measured by intrinsic fluorescence (D) release of imiquimod (IMQ) from nanoporous microneedle arrays in phosphate-buffered saline measured by high-performance liquid chromatography.

Figure 4

Representative quantification image of the delivery of fluorescently labeled antigen into mouse ears. (A,B) An overlay of picture of the mouse ear and infrared fluorescence imaging. Two independent ear piercing experiments are shown for diphtheria toxoid (DT) (n = 2) (A) and tetanus toxoid (n = 3) (B). (C,D) Background fluorescence without and with mouse ear. (E) Gradient of solution containing 0.24, 0.6, and 1.2 Lf DT. Blue circles all indicate region of interest and have an equal diameter in all cases.

Figure 5

Serum immune globulin G (IgG) responses (mean + individual results) after immunization with phosphate-buffered saline or diphtheria toxoid (DT) and tetanus toxoid (TT) intradermal (ID) loaded onto nanoporous microneedle array or SC, both routes with or without imiquimod (IMQ). When IMQ was added, only half of the antigen dose was applied. IgG responses were detected against DT antigens (A–C) and TT antigens (D–F). Kruskal–Wallis test with Dunn’s post hoc test was performed to determine statistical differences between midpoint titers determined using four different titers dilutions.

Figure 6

Ratio IgG1:IgG2a serum responses after microneedle-based intradermal (ID) and subcutaneous (SC) immunization with diphtheria toxoid (DT) (A,B) and tetanus toxoid (TT) (C,D) after the first boost (A,C) and second boost (B,D) vaccination. When antigens were adjuvanted with imiquimod (IMQ), only half of the antigen dose was applied compared with non-adjuvanted groups. Kruskal–Wallis test with Dunn’s post hoc test was performed to determine statistical differences between ratios.