Microneedle-based transcutaneous immunisation in mice with N-trimethyl chitosan adjuvanted diphtheria toxoid formulations

Abstract

Purpose: The purpose of this study was to gain insight into the delivery and immunogenicity of N-trimethyl chitosan (TMC) adjuvanted diphtheria toxoid (DT) formulations applied transcutaneously with microneedles.

Methods: Mice were vaccinated with DT-loaded TMC nanoparticles, a solution of TMC and DT (TMC/DT) or DT alone. The formulations were applied onto the skin before or after microneedle treatment with two different 300-microm-long microneedle arrays and also injected intradermally (ID). As a positive control, alum-adjuvanted DT (DT-alum) was injected subcutaneously (SC). Ex vivo confocal microscopy studies were performed with rhodamine-labelled TMC.

Results: Independent of the microneedle array used and the sequence of microneedle treatment and vaccine application, transcutaneous immunisation with the TMC/DT mixture elicited 8-fold higher IgG titres compared to the TMC nanoparticles or DT solution. The toxin-neutralising antibody titres from this group were similar to those elicited by SC DT-alum. After ID immunisation, both TMC-containing formulations induced enhanced titres compared to a DT solution. Confocal microscopy studies revealed that transport of the TMC nanoparticles across the microneedle conduits was limited compared to a TMC solution.

Conclusions: In conclusion, TMC has an adjuvant function in transcutaneous immunisation with microneedles, but only if applied in a solution.

Fig. 1

The two types of microneedles used in this study. A: Array of 300A microneedles, manufactured from commercially available 30G needles. B: Array of 300ED microneedles, made of stainless steel. Both arrays contain sixteen microneedles with a length of 300 µm. In figure C and D higher magnification images of a single 300A microneedle (C) and a 300ED microneedle (D) are shown.

Fig. 2

IgG (A) and neutralising antibody (B) titres obtained after piercing with 300A microneedles followed by application of a DT solution, a TMC/DT mixture or DT-loaded TMC nanoparticles (TMC NP) as compared to SC DT-alum. A: IgG titres after prime and two booster vaccinations. Mean and SD of 8 mice. B: Neutralising antibody titres after second boost. Data are expressed as the highest dilution that was still capable of protecting the Vero cells against challenge with diphtheria toxin. ** p < 0.01, *** p < 0.001.

Fig. 3

Effects of microneedle array type and co-administration of CT on the immunogenicity of the DT-loaded TMC nanoparticles (TMC NP) after microneedle pre-treatment. A: IgG titres after prime and 2 booster vaccinations. B: Neutralising antibody titres after 2nd boost. Data are expressed as the highest dilution that was still capable of protecting the Vero cells against challenge with diphtheria toxin. C: IgG1 and IgG2a titres after 2nd boost. A/C: Mean and SD of 8 mice. B: Individual values and geometric mean of 8 mice are shown. * p < 0.05, ** p < 0.01, *** p < 0.001.

Fig. 4

IgG (A) and neutralising antibody (B) titres after ID vaccination with the different formulations. A: IgG titres after prime and 2 booster vaccinations. B: Neutralising antibody titres after 2nd boost. Data are expressed as the highest dilution that was still capable of protecting the Vero cells against challenge with diphtheria toxin. C: IgG1 and IgG2a titres after 2nd boost. A/C: Mean and SD of 8 mice. B: Individual values and geometric mean of 8 mice are shown. • significantly higher compared to ID DT. ‡ significantly higher compared to SC DT-alum. * p < 0.05, ** p < 0.01.

Fig. 5

Representative images of microneedle conduits in mouse skin. A: x,y image (parallel to skin surface) showing 8 conduits at the skin surface. B: x,z image (perpendicular to the skin surface) showing penetration of TMC-rhodamine until a depth of approximately 150 µm.

Fig. 6

Determination of the fluorescence in the skin after microneedle pre-treatment and application of either TMC-rhodamine nanoparticles or a TMC-rhodamine solution. Area containing fluorescence plotted against the skin depth. Mean and SEM of 3 mice.

Fig. 7

X,y images (parallel to the skin surface) showing the distribution pattern in the dermis after ID injection of either a TMC-rhodamine solution (A) or TMC-rhodamine nanoparticles (B).