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Figure 1. Fluorescence microscopical images of the (a) twoand (b) three-layered FL-OA dissolving microneedles made of chondroitin sulfate as the base used in the in vivo delivery and distribution experiments. Left panels portray overview images of dissolving microneedles under normal light.

Figure 2. Fluorescence microscopy of enlarged image of the (A) twoand (B) three-layered FL-OA dissolving microneedles. Left panels portray overview images of dissolving microneedles under normal light. Arrows indicate the following lengths: (a) Whole length; (b) Length of the FL-OA space; (c) Diameter of the basement.

Table 1. Physicochemical property of twoand three-layered dissolving microneedles.

in Table 2.

In the case of three-layered OA microneedle array chips, the OA contents were, respectively, 2.2 ± 0.3, 13.7 ± 0.5, and 21.1 ± 1.6 mg. However, the recovered OA contents in the chips were, respectively, 0.5 ± 0.2, 1.0 ± 0.8, and 0.7 ± 0.3 mg. By subtracting the recovered amount of OA from the amount of OA formulated in

Table 2. OA contents in dissolving microneedle array chip, recovered amount of OA and administered dose.

microneedle array chips, the administered doses of OA were estimated respectively as 1.8 ± 0.2, 12.6 ± 0.7, and 20.4 ± 0.3 mg.

In Table 3 and Figure 3, the anti- concentrations are shown after application of twoand three-layered dissolving microneedles and sc injection of OA solution. When OA solutions were injected to the rats at three different doses of 10, 100, and 1000 mg, the anti- concentrations at 2 weeks after the first immunization were, respectively, 2.0 ± 0.8, 4.7 ± 1.1, and 9.7 ± 3.1 (×104 U/mL). At 2 weeks after the second immunization, the anti- concentrations were more increased, to 67.1 ± 23.5, 124.0 ± 38.1, and 164.0 ± 30.0 (×104 U/mL), respectively.

After application of the two-layered dissolving microneedles, the anti- concentrations at 2 weeks after the first immunization were, respectively, 3.2 ± 0.3, 2.5 ± 0.5 and 2.8 ± 0.6 (×104 U/mL). At 2 weeks after the second immunization, the anti- concentrations were, respectively, 39.8 ± 13.3, 145.6 ± 25.8, and 70.3 ± 39.2 (×104 U/mL).

Three-layered dissolving microneedles showed higher plasma levels at both 2 and 4 weeks. The anti- concentrations at 2 weeks after the first immunization were, respectively, 16.5 ± 1.9, 13.0 ± 4.3, and 14.4 ± 0.6 (×104 U/mL). At 2 weeks after the second immunization, the anti- concentrations were, respectively, 282.8 ± 78.5, 368.4 ± 42.5, and 394.6 ± 45.6 (×104 U/mL). At 4 weeks after the first administration, three-layered microneedles showed about 2.5 - 7.0 fold and 5.4 fold higher total antibody levels than either the two-layered microneedles or sc injection solution.

Using plasma samples obtained at 2 weeks after the start of the experiment and the pre-dose plasma samples,

Table 3. Administered dose of OA and total Ig(G+A+M) antibody.

Figure 3. Plasma OA specific total Ig (G + A + M) antibody concentrations at (a) 2 and (b) 4 weeks post first immunization after transcutaneous administration of twoand threelayered dissolving microneedles containing OA to the abdominal rat skin. Plasma OA specific total Ig(G + A + M) antibody concentrations were measured using ELISA. The gray bars denote antibody concentrations obtained after administration of two-layered OA dissolving microneedles, 22.0 ± 0.2 µg/rat. The black bars denote those obtained after administration of three-layered OA dissolving microneedles, 20.4 ± 0.3 µg/rat. The white bars denote those obtained after subcutaneous administration of OA: 100 µg/rat. Each value shows the mean ± S.E. of 4 - 5 experiments. *p < 0.05, **p < 0.01: significantly different from three-layered microneedles. Each value shows the mean ± S.E. (n = 4 - 5).

anti-OA IgE levels were measured. The results are shown in Table 4. The IgE concentrations, after the administration of placebo microneedles, made without OA, and two-layered microneedles (12.0 ± 0.2, 22.0 ± 0.2 mg/rat), three-layered microneedles (12.6 ± 0.7, 20.4 ± 0.3 mg/rat)

Table 4. Plasma anti-OA IgE levels and the changing rate of IgE level at 2 weeks after the start of transcutaneous immunization against the pretreatment level.

and sc, were, respectively, 50.9 ± 0.5, 56.1 ± 1.3, 59.9 ± 0.9, 58.2 ± 1.9, 56.37 ± 0.8, and 63.1 ± 1.0 (U/mL). In contrast, the IgE concentrations before immunization were, respectively, 51.9 ± 0.9, 58.9 ± 0.2, 57.4 ± 1.9, 61.7 ± 0.6, 60.2 ± 1.1, and 53.9 ± 1.0 (U/mL). In addition, the estimated rates of change of IgE level were, respectively, –1.7% ± 2.3%, –4.7% ± 2.0%, 5.8% ± 1.9%, –5.4% ± 4.0%, –6.3% ± 2.5%, and 17.0% ± 1.6%. No significant difference was found in the rate of change of anti-OA IgE level between placebo, two-layered and threelayered groups, although the sc injection group showed a significantly higher level than the placebo group.

3.3. Delivery and Distribution Study in Rat Skin

The delivery site and the diffusion characteristics of the vaccine antigen in the rat skin were studied by administering the twoor three-layered microneedles containing FL-OA used as a model antigen.

Figure 4 portrays normal and fluorescent images of rat skin sections obtained after the administration of FL-OA loaded dissolving microneedles to the rat skin. In the case of two-layered microneedles containing FL-OA, the spots were detected around the first 200 mm of rat skin. However, the spots of green fluorescein, which had been delivered from three-layered microneedles containing FL-OA, were detected mainly from the surface to the first 100 mm of the skin. As the figures show, the green fluorescence derived from FL-OA was apparent immediately after its administration; it diffused as time passed.

Because chondroitin sulfate, a water-soluble threadforming polymer, was used as the base polymer to prepare dissolving microneedles, the base dissolved rapidly. Consequently, the release of green fluorescein occurred immediately after administration. Even at 30 s after administration, the conical shape of dissolving microneedles was not detected completely, although spots of green fluorescein were detected. At 2 and 5 min after administration, green fluorescence spots enlarged transversally. Then diffusion to the transverse direction reached the steady state at 10 min.

Figure 5 presents the horizontal distribution profiles of fluorescent intensity attributable to FL-OA delivered in the rat skin after administration by dissolving microneedles. In the case of two-layered microneedles containing FL-OA, the maximum fluorescent intensity was detected at the first 120 mm of rat skin. The distribution profile showed a gradual increase in fluorescence intensity with an increase in the depth from surface to 120 mm, and a gradual decrease in it with an increase deep from 120 mm onward. Furthermore, at the depth of 160 - 260 mm, the fluorescence intensity distribution was considerably higher than that obtained from three-layered microneedles. The maximum fluorescence intensity obtained after the administration of three-layered microneedles was detected at the first 20 mm of the skin. The fluorescence intensity decreased gradually with increased depth. At this depth, distribution of fluorescence intensity was markedly higher than that obtained using two-layered microneedles. The figures show that the twoand three-layered microneedles respectively delivered the model vaccine antigen mainly to the dermal and epidermal layers.

4. Discussion

The main function of skin is to provide a protective cover against the hostile external environment: a role that includes immunoprotection. Human skin comprises three layers: the stratum corneum, epidermis, and dermis. The first one is the 10 - 15 μm thick outer layer, which is dead tissue. The stratum corneum is a strong primary barrier against exogenous compounds, including drugs. The second barrier is the viable epidermis, 100 - 150 μm, which contains tissues such as living cells. However, the epidermis has no blood vessels. The epidermis primarily comprises keratinocytes, but it also has, distributed amongst the viable keratinocytes, a large population of cells, i.e. 1% - 3% of epidermal cells [28], that are involved in immune surveillance, the LCs. Although LCs

Figure 4. Fluorescence microscopy of the skin of rats who received the (a) twoand (b) three-layered dissolving microneedles containing FL-OA, as visualized through a 50× objective (scale bar: 200 μm). Dissolving microneedles of two types were administered to the rat abdominal skin. Thereafter, skin tissue samples were obtained at 30 s and 2, 5, and 10 min. Right panels show the corresponding brightfield microscopy images.

Figure 5. Distribution profiles of FL-OA in the rat skin after administration by dissolving microneedles. Dissolving microneedles of two types were administered to the rat skin. Skin tissue samples were obtained at 1 min. The 20 μm thick slice samples were obtained and FL-OA contents in each slice were determined spectrofluorometrically. The distribution ratio of FL-OA in rat skin was calculated using 100× fluorescent intensity of each section/maximum fluorescent intensity in all sections. The open squares show data obtained after administration of two-layered dissolving microneedles containing FL-OA. The open circles represent data for three-layered dissolving microneedles. **p < 0.01; significantly different from two-layered microneedles. *p < 0.05; significantly different from three-layered microneedles. Each value shows the mean ± S.E. (n = 5).

are few among the cells in the skin, they account for 25% of the total skin surface area in humans [29]. In fact, LCs represent an extensive, superficial network barrier of immune cells that make an attractive target for vaccine delivery. The LCs are bone marrow-derived dendritic cells that migrate to epithelial surfaces such as the skin where they perform immunosurveillance [30]. Under normal circumstances, the baseline traffic of LCs exists from the skin to the draining lymph nodes, where the cells present the antigens they have encountered in the epidermis. To increase the skin permeability of drugs, numerous approaches have been attempted using chemical enhancers, electric fields, ultrasound, and thermal methods [6-10]. However, these TDDS’ success has been limited because of the strong barrier function of the skin: the low membrane permeability of drugs through the skin. In our earlier studies, two-layered dissolving microneedles with water-soluble thread-forming biopolymers such as chondroitin sulfate, dextran, hyaluronic acid and albumin used as the base were evaluated as a new TDDS. The drug was formulated as a solid dispersion. After administration to the rat skin, high BAs of 91.3% - 97.7% were obtained for insulin in mice [14] and of 81.5% - 102.3% for LMWH in rats [15]. Furthermore, high BAs of 87.5% were obtained for rhGH in rats [16] and of 82.1% - 99.4% for EPO in mice [17]. The relative BA of IFN against sc injection of IFN solution was 79.9% - 117.8% in rats [18]. The relative BA of insulin was 90% - 99% in dogs [19]. Based on those outcomes, dissolving microneedles have been applied for transcutaneous administration of vaccine antigen. In previous studies of transcutaneous immunization, the mouse model system has been used because many immunological assay systems have been developed and are now commercially available. However, the mouse model is not useful for the study of transcutaneoous immunization, a so-called skin vaccine, because the histology of mouse and human skin differs considerably. According to a report by Monteiro-Riviere et al., the epidermal thickness at the skin of the ventral abdomen is 11.58 ± 1.02 µm for rats and 22.47 ± 2.40 µm for dogs [31]. For human skin, the epidermal thickness is reportedly to be 60.3 ± 15.0 µm [32]. It is very difficult to deliver vaccine antigen strictly relying upon the epidermis and/or dermis for any delivery system in mice. Therefore, in this study, we introduced a rat model system. In this study, we used Brown Norway rats because Brown Norway rats have higher sensitivity to OA than Hooded Lister, Piebald Virol Glaxo, and Wistar rats have [33]. In the case of abdominal skin in rats, the respective thicknesses of the stratum corneum, epidermis, and dermis have been reported as 4.56 ± 0.61 µm, 11.58 ± 1.02 µm, and 402.0 ± 49.86 µm [31,34]. Although a difference exists in the thickness and histology of the skin between rats and humans, it is possible to deliver vaccine antigen to the epidermis and/or dermis using dissolving microneedle array chips. Gelinck et al. used an intradermal injection syringe (BD Micro-fine 0.5 mL U-100 insulin syringe; Microfine Materials Technologies Pte. Ltd.) to administer influenza vaccine antigen to human subjects [35]. However, it is difficult to control the depth of the injection site of vaccine antigen solution inside the skin. Therefore, to perform this study, we designed dissolving microneedles of two types: two-layered and three-layered dissolving microneedles. In the case of two-layered microneedles, OA, the model antigen, was formulated in the acral portion of microneedles, but OA was formulated into the second layer of three-layered microneedles. For two-layered microneedles, the length of the OA formulated layer was 155 ± 5 µm from the top of the microneedles. In the case of three-layered microneedles, OA was formulated at the position of 175 ± 4 μm to 225 ± 5 µm from the top of the microneedles. The epidermal thickness was reported as 11.58 ± 1.02 µm in rats and the layer of dermis is 402.0 ± 49.86 µm [31,34]. Consequently, OA would be delivered to the subcutaneous tissue of the rat if the microneedle of 500 µm length were inserted completely into the rat skin. However, the full length of the microneedles was not inserted into the skin, as our studies of dissolving microneedles conducted for over a decade have confirmed. Our previous report showed that about a half-length of the microneedles was inserted into the skin [36]. Furthermore, when we inserted 500 µm length dissolving microneedles into the skin, no bleeding occurred. Those results suggested that the acral portion of dissolving microneedles was dissolved during passage through the skin. The width of the top portion of dissolving microneedles was approximately 10 µm. When the acral portion of dissolving microneedles was inserted into the skin, the dissolving microneedles encountered environmental water and dissolved during passage through the epidermis, because Caspers et al. reported that the water content in the skin epidermal layer was as high as 70% [37]. Fluorescent microscopical experiments using two-layered and threelayered dissolving microneedles containing FL-OA in this study showed that two-layered microneedles delivered FL-OA mainly to the rat skin dermal layer, where dermal DCs are highly distributed, and three-layered microneedles delivered FL-OA mainly to the epidermal layer where LCs are highly distributed. Some controversy persists about the target cells of the transcutaneously delivered antigen. Several groups support LCs. Other groups support dermal DCs. However, this argument remains unresolved, because no device can deliver vaccine antigen to a specific site of the skin, dermis and/or epidermis. Therefore, twoand three-layered dissolving microneedles were prepared. These devices delivered vaccine antigen strictly to the dermis and epidermis. Before our study of transcutaneous immunization, coated microneedle array chips were applied to study the effect of delivery parameters on immunization to OA that was coated onto the surface of microneedles [38]. For the coated microneedles, the distribution of OA in the rat skin and the antibody responses were independent of the microneedle length used for the delivery of OA, antigen. Moreover, it was shown to be very difficult to control the coating length from the top of the microneedles. Furthermore, in the case of coating microneedles, a vaccine antigen was coated onto the surface of the acral portion of microneedles. In this case, the amount of antigen was limited. In other words, the coating amount of vaccine antigen on the acral surface of microneedles, which was made of steel or a biopolymer-like polylactic acid, is limited. When steel microneedles are used as the device to administer vaccine transcutaneously, a problem exists in terms of its safety: allergy to the steel. To study the safety of OA-loaded dissolving microneedles, plasma IgE levels were measured at 2 weeks after the start of immunization. In addition, a histological study of the skin tissue was performed after the administration of microneedle array chips. Results showed no significant difference in the rate of increase of IgE levels between active groups and control groups who received placebo microneedles. In our previous studies of the evaluation of the dissolving microneedles containing insulin, EPO and rhGH, no irritation or damage was found at the administered skin tissue at 2 hr and 24 hr after administration [20, 22]. In this study, the rat skin was monitored for 1 month. However, no irritation or damage was found in the skin tissue over that long term of 1 month. Therefore, we infer that dissolving microneedles are a safe transcutaneous drug delivery system for use in immunization.

5. Conclusion

This feasibility study of OA loaded twoand three-layered dissolving microneedles having 500 μm length and 300 μm diameters of their basements was performed using rats. The dissolving microneedles were prepared with chondroitin sulfate as the base polymer using microfabrication technology. The delivery of model antigen, OA, to the epidermis and dermis was ascertained using threelayered and two-layered dissolving microneedles, where OA was formulated respectively as a solid dispersion with chondroitin sulfate at the second portion, 175 ± 4 - 225 ± 5 μm, and acral portion, 0 - 155 ± 5 μm, of microneedles from their tops. Systemic immune responses, OA-specific antibody in plasma, were 2.5 - 7 fold increased by three-layered dissolving microneedles compared to two-layered dissolving microneedles. Histological studies using FL-OA loaded dissolving microneedles revealed that FL-OA was delivered mainly to the epidermal region of the rat skin, around the first 100 μm of the skin, by the three-layered dissolving microneedles. Those results revealed that the delivery site of transcutaneous antigen by three-layered dissolving microneedles was the epidermis of the skin where LCs are highly distributed.

6. Acknowledgements

This study was supported by a Grant-in-Aid for scientific Research (b) (223100820001) from Ministry of Education, Science, Sports and Culture of Japan, MEXT. This study was also supported by a strategic fund of MEXT from 2008 to 2013 for establishing research foundation in private universities of Japan.

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