Figure 1. Bacteriostatic properties of the microneedle matrix. (a–j) Bacterial colonies formed in microneedle matrix solutions with E. coli added by agar dilution method. (a’–j’) Bacterial colonies formed in microneedle matrix solutions stored at room temperature (RT) for 14 d by agar dilution method. Samples with fish gelatin are outlined in red boxes. Microneedle matrix solutions: (a,a’) sterile water, (b,b’) 1% BSA, (c,c’) 10% sucrose, (d,d’) 35% nano hyaluronic acid + 1% BSA, (e,e’) 5% PVA + 1% BSA (10-fold dilution), (f,f’) 5% PVP-k17 + 1% BSA, (g,g’) 35% fish gelatin, (h,h’) 35% fish gelatin + 10% sucrose, (i,i’) 35% fish gelatin + 10% sucrose + 1% BSA, (j,j’) 10% fish gelatin + 10% sucrose + 1% BSA.

Figure 1. Bacteriostatic properties of the microneedle matrix. (a–j) Bacterial colonies formed in microneedle matrix solutions with E. coli added by agar dilution method. (a’–j’) Bacterial colonies formed in microneedle matrix solutions stored at room temperature (RT) for 14 d by agar dilution method. Samples with fish gelatin are outlined in red boxes. Microneedle matrix solutions: (a,a’) sterile water, (b,b’) 1% BSA, (c,c’) 10% sucrose, (d,d’) 35% nano hyaluronic acid + 1% BSA, (e,e’) 5% PVA + 1% BSA (10-fold dilution), (f,f’) 5% PVP-k17 + 1% BSA, (g,g’) 35% fish gelatin, (h,h’) 35% fish gelatin + 10% sucrose, (i,i’) 35% fish gelatin + 10% sucrose + 1% BSA, (j,j’) 10% fish gelatin + 10% sucrose + 1% BSA.

Figure 2. Preparation and characterization of the microneedle patch. (a) Diagram of the preparation process of the microneedle patch. (b) The silicone microneedle mold used to prepare the microneedle patches. (c) Overall view of the dissolving microneedle patch. (d) View of the microneedle array (10 × 10, 100 needles per patch) under stereoscopic microscope. Electron microscopy giving: (e) side view of microneedles (needle length of 650 μm) and (f) top view of microneedles (bottom surface diameter of 360 μm).

Figure 2. Preparation and characterization of the microneedle patch. (a) Diagram of the preparation process of the microneedle patch. (b) The silicone microneedle mold used to prepare the microneedle patches. (c) Overall view of the dissolving microneedle patch. (d) View of the microneedle array (10 × 10, 100 needles per patch) under stereoscopic microscope. Electron microscopy giving: (e) side view of microneedles (needle length of 650 μm) and (f) top view of microneedles (bottom surface diameter of 360 μm).

Figure 3. Mechanical performance of microneedle patches and their penetration and dissolving in the skin. (a) Diagram of the displacement-force test station. (b) Displacement-force for single microneedles (mean ± SEM, n = 5, one-way ANOVA, p > 0.05). Different color curves represent microneedle patches with different matrix materials. The horizontal axis is the distance the force gauge probe moves downward after contacting the microneedle, and the vertical axis is the pressure applied to each microneedle. (c) Displacement-force produced with microneedle patches (100 microneedles per patch, mean ± SEM, n = 5, one-way ANOVA, p > 0.05). (d) Microneedle patch before and after 20 N pressure applied. The red circle shows the microneedle that was only slightly deformed by the pressure of 20 N. (e) Pathological section of mouse skin before and after microneedle patch penetration. The red circle shows the area where the microneedle penetrated. (f) OCT scans of mouse skin and pig skin before and after microneedle patch penetration. (g) CLSM images of microneedle patches after penetration of mouse skin for 0–15 min and penetration of pig skin for 0–5 min. The red parts represent microneedles containing sulforhodamine B.

Figure 3. Mechanical performance of microneedle patches and their penetration and dissolving in the skin. (a) Diagram of the displacement-force test station. (b) Displacement-force for single microneedles (mean ± SEM, n = 5, one-way ANOVA, p > 0.05). Different color curves represent microneedle patches with different matrix materials. The horizontal axis is the distance the force gauge probe moves downward after contacting the microneedle, and the vertical axis is the pressure applied to each microneedle. (c) Displacement-force produced with microneedle patches (100 microneedles per patch, mean ± SEM, n = 5, one-way ANOVA, p > 0.05). (d) Microneedle patch before and after 20 N pressure applied. The red circle shows the microneedle that was only slightly deformed by the pressure of 20 N. (e) Pathological section of mouse skin before and after microneedle patch penetration. The red circle shows the area where the microneedle penetrated. (f) OCT scans of mouse skin and pig skin before and after microneedle patch penetration. (g) CLSM images of microneedle patches after penetration of mouse skin for 0–15 min and penetration of pig skin for 0–5 min. The red parts represent microneedles containing sulforhodamine B.

Figure 4. Immunogenicity and protective efficacy of microneedle vaccines. (a–c) Serum IgG antibody titers after AHc vaccination of mice (mean ± SEM, n = 5, t-test, p > 0.05, NS: no significant difference). The horizontal axis indicates the number of vaccinations. The vertical axis is the serum IgG antibody titers of mice. The doses of AHc vaccination were 20 μg, 2 μg, and 0.2 μg in (a–c), respectively. (a’–c’) Survival curves of mice after protective efficacy assay of 106 LD50 BoNT/A. The horizontal axis is time. The vertical axis is the survival percentage of mice. Doses of AHc vaccination were 20 μg, 2 μg, and 0.2 μg in (a’–c’), respectively.

Figure 4. Immunogenicity and protective efficacy of microneedle vaccines. (a–c) Serum IgG antibody titers after AHc vaccination of mice (mean ± SEM, n = 5, t-test, p > 0.05, NS: no significant difference). The horizontal axis indicates the number of vaccinations. The vertical axis is the serum IgG antibody titers of mice. The doses of AHc vaccination were 20 μg, 2 μg, and 0.2 μg in (a–c), respectively. (a’–c’) Survival curves of mice after protective efficacy assay of 106 LD50 BoNT/A. The horizontal axis is time. The vertical axis is the survival percentage of mice. Doses of AHc vaccination were 20 μg, 2 μg, and 0.2 μg in (a’–c’), respectively.

Figure 5. Stability of the microneedle vaccine. (a) Serum IgG antibody levels of mice after vaccination with AHc (0.2 μg per mouse) stored at room temperature. The horizontal axis is time of AHc storage. The vertical axis is the ratio of serum OD of mice after the last vaccination to the OD of the negative control (mean ± SEM, n = 5, t-test, NS: no significant difference, ****: p < 0.0001, ***: p < 0.0002). (b) Survival of mice after protective efficacy assay of 106 LD50 BoNT/A. The horizontal axis is the time of AHc storage at room temperature. The vertical axis is the survival percentage of mice.

Figure 5. Stability of the microneedle vaccine. (a) Serum IgG antibody levels of mice after vaccination with AHc (0.2 μg per mouse) stored at room temperature. The horizontal axis is time of AHc storage. The vertical axis is the ratio of serum OD of mice after the last vaccination to the OD of the negative control (mean ± SEM, n = 5, t-test, NS: no significant difference, ****: p < 0.0001, ***: p < 0.0002). (b) Survival of mice after protective efficacy assay of 106 LD50 BoNT/A. The horizontal axis is the time of AHc storage at room temperature. The vertical axis is the survival percentage of mice.

Table 1. Formulation of microneedle matrix solutions.

Table 1. Formulation of microneedle matrix solutions.

No.MatrixConcentration
(w/v)Excipient10% Sucrose1% BSA1Fish gelatin35%−−2Fish gelatin35%+−3Fish gelatin35%++4Fish gelatin10%++5Nano hyaluronic acid35%−+6PVA5%−+7PVP5%−+8Sterile water −−9Sterile water+−10Sterile water−+

Table 2. Vaccination experiment groups.

Table 2. Vaccination experiment groups.

No.Vaccination MethodDosage (µg)Number of Mice per Group1MN20152MN2153MN0.2154MN0155SC20156SC2157SC0.2158SC015