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. 2017 Jul 4:12:4763-4772.
doi: 10.2147/IJN.S132456. eCollection 2017.

Enhanced immunization via dissolving microneedle array-based delivery system incorporating subunit vaccine and saponin adjuvant

Ji-Hui Zhao  1 , Qi-Bo Zhang  1 , Bao Liu  2 , Xiang-Hua Piao  1 , Yu-Lu Yan  1 , Xiao-Ge Hu  1 , Kuan Zhou  1 , Yong-Tai Zhang  1 , Nian-Ping Feng  1
Affiliations

Enhanced immunization via dissolving microneedle array-based delivery system incorporating subunit vaccine and saponin adjuvant

Ji-Hui Zhao et al. Int J Nanomedicine. .

Abstract

Purpose: To enhance the immunogenicity of the model subunit vaccine, ovalbumin (OVA) was combined with platycodin (PD), a saponin adjuvant. To reduce the toxicity of PD, OVA, and adjuvant were loaded together into liposomes before being incorporated into a dissolving microneedle array.

Methods: OVA- and PD-loaded liposomes (OVA-PD-Lipos) were prepared using the film dispersion method. Their uptake behavior, toxicity to mouse bone marrow dendritic cells (BMDCs), and hemolytic activity to rabbit red blood cells (RBCs) were evaluated. The OVA-PD-Lipos were incorporated into a dissolving microneedle array. The chemical stability of OVA and the physical stability of OVA-PD-Lipos in microneedle arrays were investigated. The immune response of Institute of Cancer Research mice and potential skin irritation reaction of rabbits to OVA-PD-Lipos-MNs were evaluated.

Results: The uptake of OVA by mouse BMDCs was greatly enhanced when OVA was prepared as OVA-PD-Lipos, and in this form, the toxicity of PD was dramatically reduced. OVA was chemically stable as OVA-PD-Lipos, when OVA-PD-Lipos was incorporated into a dissolving microneedle array. Institute of Cancer Research mice treated with OVA-PD-Lipos-MNs showed a significantly enhanced immune response. PD combined with OVA elicited a balanced Th1 and Th2 humoral immune response in mice, with minimal irritation in rabbit skin.

Conclusion: The dissolving microneedle array-based system is a promising delivery vehicle for subunit vaccine and its adjuvant.

Keywords: dissolving microneedle array; intradermal vaccination; liposomes; saponin adjuvant; subunit vaccine.

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Conflict of interest statement

Disclosure The authors report no conflicts of interest in this work.

Figures

Figure 1
Figure 1
Hemolytic activity of OVA-PD-Lipos in rabbit RBCs (n=3). Abbreviations: OVA, ovalbumin; PD, Platycodin D; RBCs, red blood cells.
Figure 2
Figure 2
Toxicity of OVA-PD-Lipos to cultured mouse BMDCs (n=6). Abbreviations: BMDCs, bone marrow dendritic cells; OVA, ovalbumin; PD, Platycodin D.
Figure 3
Figure 3
Uptake of FITC-OVA-PD-Lipos and FITC-OVA by cultured mouse BMDCs as determined by flow the units for the y-axes of this figure are cell count. Abbreviations: BMDCs, bone marrow dendritic cells; FITC, fluorescein isothiocyanate; PD, Platycodin D; OVA, ovalbumin.
Figure 4
Figure 4
Transmission electron micrographs of (A) empty liposomes, (B) OVA-PD-Lipos, and (C) OVA-PD-Lipos-MNs. Note: TEM was performed after OVA-PD-Lipos-MNs were dissolved in distilled water. Abbreviations: OVA, ovalbumin; PD, Platycodin D; MNs, microneedle array.
Figure 5
Figure 5
The integrity of OVA in OVA-PD-Lipos-MNs analyzed by SDS-PAGE. Notes: Lane (A) protein molecular mass markers; Lane (B) OVA standard; Lane (C) OVA-PD-Lipos; Lane (D) OVA-PD-Lipos-MNs. Abbreviations: OVA, ovalbumin; PD, Platycodin D; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; MNs, microneedle array.
Figure 6
Figure 6
Evaluation of OVA-PD-Lipos-MNs inserted into mouse skin in vitro. Notes: (A) Array of needle holes in mouse skin stained with trypan blue after insertion. (B and C) Vertical slices of mouse skin before and after insertion of OVA-PD-Lipos-MNs (stained using H&E). Abbreviations: H&E, hematoxylin–eosin; OVA, ovalbumin; PD, Platycodin D; MNs, microneedle array.
Figure 7
Figure 7
Images of nude mice after topical application of (A) FITC-OVA-PD-Lipos and (B) FITC-OVA-PD-Lipos-MNs. The colored area represents fluorescent intensity. Abbreviations: FITC, fluorescein isothiocyanate; OVA, ovalbumin; PD, Platycodin D; MNs, microneedle array.
Figure 8
Figure 8
Dissolution of OVA-PD-Lipos-MNs after insertion into mouse skin in vivo. Abbreviations: OVA, ovalbumin; PD, Platycodin D; MNs, microneedle array.
Figure 9
Figure 9
OVA-specific IgG, IgG1 and IgG2b levels in ICR mice immunized with OVA-PD-Lipos-MNs (n=6). Notes: OVA, OVA + PD, and OVA-PD-Lipos represent OVA solution, OVA and PD solution, OVA and PD loaded liposomes, respectively; OVA-MNs, OVA-PD-MNs, and OVA-PD-Lipos-MNs represent free OVA, free OVA + PD, and OVA-PD-Lipos, loaded into dissolving microneedle array, separately; no statistical difference was found among groups in the same box; *P<0.05. Abbreviations: OVA, ovalbumin; PD, Platycodin D; MNs, microneedle array.
Figure 10
Figure 10
Response of intact rabbit skin after multiple-dose delivery of OVA-PD-Lipos-MNs. Notes: Rows A–D represent blank control, blank dissolving microneedles, free OVA and PD in dissolving microneedles, and OVA-PD-Lipos-MNs, respectively. Column W1-1 h and W2-1 h represent 1 h after the washing out of the first and second dose; Column W3-1 h, W3-24 h, W3-48 h, and W3-72 h represent 1, 24, 48, and 72 h after the washing out of the third dose, respectively. Column T2-0 h and T3-0 h represent immediately before the application of the second and third dose. Abbreviations: OVA, ovalbumin; PD, Platycodin D; MNs, microneedle array.
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