BCG vaccine powder-laden and dissolvable microneedle arrays for lesion-free vaccination

Abstract

Live attenuated Bacille Calmette-Guerin (BCG) bacillus is the only licensed vaccine for tuberculosis prevention worldwide to date. It must be delivered intradermally to be effective, which causes severe skin inflammation and sometimes, permanent scars. To minimize the side effects, we developed a novel microneedle array (MNA) that could deliver live attenuated freeze-dried BCG powder into the epidermis in a painless, lesion-free, and self-applicable fashion. The MNA was fabricated with biocompatible and dissolvable hyaluronic acid with a deep cave formed in the basal portion of each microneedle, into which BCG powder could be packaged directly. Viability of BCG vaccine packaged in the caves and the mechanical strength of the powder-laden MNA did not alter significantly before and after more than two months of storage at room temperature. Following insertion of the MNA into the skin, individual microneedle shafts melted away by interstitial fluid from the epidermis and upper dermis, exposing the powder to epidermal tissues. The powder sucked interstitial fluid, dissolved slowly, and diffused into the epidermis in a day against the interstitial fluid influx. Vaccination with BCG-MNA caused no overt skin irritation, in marked contrast to intradermal vaccination that provoked severe inflammation and bruise. While causing little skin irritation, vaccination efficacy of BCG-MNAs was comparable to that of intradermal immunization whether it was evaluated by humoral or cellular immunity. This powder-laden and dissolvable MNA represents a novel technology to sufficiently deliver live attenuated vaccine powders into the skin.

Keywords: BCG powder; Lesion-free; Microneedles; Skin delivery.

Figure 1.. Dissolvable and powder-laden MNAs.

Aportion of an array of dissolvable and powder-laden MNA is shown in (A). One of microneedles in A is enlarged to show the cave (B). The MNA was loaded with Alexa fluor 555-OVApow dcr (red), whereas FITC was embedded in shaft of the MNA (green) (C). Arrows in B&C indicate the cave in a microneedle. After insertion of the OVA-MNA into the ear of C57BL/6 mice (D) or MHC II-EGFPmice (E), OVA powder diffused into the skin over time (D). Three-D images (upper) and top views (lower) of one representative microneedle were obtained by two photon confocalmicroscopy at indicated times after insertion of the MNA into the skin (D). Circles in the lower panel indicate the size and location of the microoneedle. MN, microneedle. MHC II-GFP-labeled Langerhans cells in the epidermis (green) and OVA powder (red) were shown by a 3D image collected in 6 hr (upper) after OVA-MNA insertion or by a top view captured in 15 min (lower) after MNA removal (E). The ear of MHC II-EGFP transgenic mice was analyzed by confocal microscopy in 6 hr after removal of BCG-laden MNA (F).A distance between two microneedles is estimated by 4 times of the basal diameter of each microneedle (4 x bases).

Figure 2.. Characterization of BCG-MNAs.

A. A standard curve of fluorescent absorbencies at 640nm vs. varying amounts of SRB-BCG mixture. B. The amount of BCG powder within the MNA was evaluated in the basis of the standard curve in A Each symbol represents the amount of BCG in nine microneedles cut from each patch and the horizontal lines are the means of 6 patches with 95% CI (confidence interval). Results of three independent experiments designated as 1, 2, and 3 were shown each with 6 MNAs and 9 microneedles in each MNA. C. A shelf-life of BCG-MNAs was determined on day 0, 30, 60, and 90 after MNA fabrication and storage at room temperature and compared with pre-loaded BCG vaccine. Each symbol represents the number of live bacilli or colony forming units (CFUs) per 24 microneedles and the short horizontal lines are the means of 6 patches. NS, no significant difference. Statistical significance was analyzed by ANOVA D.BCG-MNA could pierce into the skin of C57BL/6 mice efficiently after 90 days of storage in room temperature, as suggested by generation of an array of micropoles in the skin with the relevant area enlarged (lower panel). E. Histological skin sections were prepared 2 hr after insertion of a BCG-MNA stored for 90 days in room temperature to show skin penetration of the individual microneedles (arrows), one of which is enlarged.

Figure 3.. Reactogenicity at the site of vaccine inoculation.

A. Photos were taken in 1, 3, 5 and 10 days after inoculation. Only areas of inflammatory and abnormal skins were outlined by a white-dot line in all panels. B. Cross-sections of the inoculation site were H&E stained 3 days after inoculation and scanned by Nanozoomer; a red circle indicates an area of inflammation induced by BCG in the skin and arrows point the sites of a hypodermic needle or microneedle insertion. Scale bar, 200 μm. C. Differences of skin surface temperature between the inoculation site and a distant area were recorded daily for 10 days (n=6). Statistical significance was analyzed by ANOVA, *P<0.05,**P<0.01, and ***P<0.001 in the presence or absence of BCG.

Figure 4.. Intracellular cytokine productions in NK and T cells.

IL-2 (A) and TNF-α (B) production by CD49B⁺ cells were assessed in the blood samples one week after vaccination. IFN-γ⁺CD4⁺ cells (C) and IFN-γ⁺CD8⁺ cells (D), and TNF-α⁺CD4⁺ cells (E) and TNF-α⁺CD8⁺ cells (F) were analyzed in blood samples four weeks post-vaccination. Data are presented as mean±SEM (n=6). Statistical significance was analysed by ANOVA *P<0.05 and NS, no significance. All experiments were repeated twice with similar results.

Figure 5.. Similar humoral responses induced by BCG delivered via either MNA or ID.

Relative IgG (A), IgGl (B) and IgG2a (C) titers in serum were measured by ELISA 4 weeks after immunization. The data are representative of two independent studies with similar results and expressed as means ± SEM (n =6). Statistical significance was analyzed by ANOVA (n=6 for each experiment). *P<0.05, ***P<0.001, and NS, no significance in the presence or absence of BCG.

Figure 6.. Representative of cytokine productions in spleen (upper) and lung (lower) after immunization.

Lymphocytes isolated from spleens (A-D) and lungs (E-H) were stimulated in vitro for72 h with mycobacterial recombinant proteins and peptides. TNF-α, IL-6, IL-12, and IL-17 were quantified in the culture supernatants by ELISA The data are representative of two independent studies with similar results and expressed as means ± SEM (n =6 for each experiment); *P<0.05, **P<0.01, ***P<0.001, and NS, no significance.