Two-and Three-Layered Dissolving Microneedles for Transcutaneous Delivery of Model Vaccine Antigen in Rats ()
1. Introduction
Percutaneous route is an attractive route for the delivery of biopharmaceuticals, because there are many advantages: 1) little or no degradation by hydrolytic enzymes compared to that in the gastrointestinal tract; 2) no firstpass effects of the liver associated with oral delivery; 3) less or no pain compared to subcutaneous injection; 4) better convenience of administration than intravenous injection; 5) a better and more continuously controlled delivery rate than those of oral and subcutaneous sustainedrelease preparations; and 6) easy removal if side-effects appear [1,2]. However, transdermal drug delivery systems (TDDSs) are not recognized as a conventional dosage form, because the permeability of drugs through the human skin is limited by the strong barrier function of the skin. Especially, biopharmaceuticals do not permeate through the skin at therapeutically relevant amounts and rates [3]. Vaccine antigens, which are also representative biopharmaceuticals, are used as an injection preparation. To develop an alternative dosage form, not only oral vaccines [4], but also skin-administered vaccines have been sought [5].
Human skin is composed of three layers: the stratum corneum, epidermis, and dermis [1]. The first one is the 10 - 15 μm thick outer layer, which is dead tissue. The second one is the stratum corneum having a strong primary barrier function against exogenous compounds, including drugs. The third barrier is the viable epidermis having the thickness of 100 - 150 μm which contains tissues such as living cells. However, there is no blood vessel in the epidermis. Several approaches such as chemical enhancers, electric fields, ultrasound, and thermal methods have been applied to increase the skin permeability of drugs, [6-10]. In the case of skin vaccine, microparticle systems are studied [11]. However, these TDDSs have not been recognized as a general TDDS, because of the strong barrier function of the skin, which prevents the permeation of drugs through the skin. The application of nanotechnology made it possible to produce microneedles by which the skin permeability of drugs was dramatically increased. Among them, coated microneedle arrays were shown to increase the transdermal permeability of ovalbumin (OA), a model antigen [12]. In addition, Matriano et al. demonstrated that skin immunization was effective by the application of antigen cream after the skin was treated with microneedles, thereby forming microconduits on the skin [13].
Those microneedles for percutaneous DDS are classified into three categories: 1) extremely small needles through which a drug solution can be injected into the skin; 2) metallic and/or silastic microneedles onto which a surface drug is coated; and 3) metallic and/or silastic microneedles which form microconduits on the skin, after which a drug solution is applied following removal of the microneedles. These materials—silicon and metals —are not optimal because they are exogenous substances. In contrast to those microneedles, we designed dissolving microneedles with water-soluble thread-forming biopolymers where chondroitin sulfate, dextran, hyaluronic acid, and albumin were used as the base. The drug was formulated as a solid dispersion. As the first step of our research, pen-type dissolving microneedle having 1 - 2 mm length and 0.4 mm diameter were prepared and inserted into the skin. In this case, the base immediately dissolved and the drug was released and absorbed into systemic circulation with high absorption efficiency. By means of dissolving microneedles, high bioavailabilities (BAs) of biopharmaceuticals were obtained, 91.3% - 97.7%, of insulin in mice [14] and 81.5% - 102.3% of low molecular weight heparin (LMWH) in rats [15]. Furthermore, high BAs of 87.5% were obtained for rhGH in rats [16] and of 82.1% - 99.4% for erythropoietin (EPO) in mice [17]. The relative BA of interferon (IFN) against subcutaneous (sc) injection of IFN solution was 79.9% - 17.8% in rats [18]. The relative BA of insulin was 90% - 99% in dogs [19]. As the second step of our research, we designed a dissolving microneedle array chip, approximately 1.0 cm2, on which 100 - 225 dissolving microneedles were formed [20]. Each dissolving microneedle had 500 mm length, with 300 mm diameter at its base. The drug was formulated at the acral portion of the dissolving microneedle, which was inserted into the skin region of the epidermis and epidermal-dermal junctions by finger pressure. Proof-of-concept experiments using insulin [20], erythropoietin [21], desmopressin [22], and recombinant human growth hormone (rhGH) [22] showed respectively high BAs: 97.9% ± 17.3%, 65.9% - 69.0%, 90.0% - 93.1%, and 72.8% - 89.9%. As a subsequent application of dissolving microneedle array chips, OA was used as a model skin vaccine antigen. The skin is densely populated with antigen presenting cells (APCs). Langerhans cells (LCs) and dermal dendritic cells are highly distributed respectively in the epidermis and dermis. Their function is to detect the environment, process antigens and present specific epitopes to T cells. Spontaneously, the dissolving microneedles physically break the barrier function of the skin and deliver the antigen to the skin tissue. However, controversy persists about the antigen target cells. Several groups regard LCs as the target [23- 25]; other groups see dendritic cells as targets [26,27]. Therefore, twoand three-layered dissolving microneedles were prepared, where OA was formulated at the acral portion of microneedles and at the second portion of microneedles from their tops, respectively. Comparison of the skin vaccination efficiency results is described in this report.
2. Materials and Methods
2.1. Materials
Ovalbumin (OA) was obtained from Sigma-Aldrich Corp. (St. Louis, MO, USA). Fluorescein labeled OA (FL-OA) was obtained from Molecular Probes Inc. (Eugene, USA). ELISA kits for measuring the OA-specific and IgE antibody concentrations in the rat plasma were obtained from Alpha Diagnostic International Inc. (TX, U.S.A.). An ELISA kit for measuring the OA contents in dissolving microneedles was obtained from Morinaga Institute of Biological Science Inc. (Tokyo, Japan). Sodium chondroitin sulfate was obtained from Nacalai Tesque Inc. (Kyoto, Japan). Brilliant blue (BB) was obtained from Tokyo Chemical Industry Co. Ltd. (Tokyo, Japan). All other materials were of reagent grade and were used as received. Male Brown Norway rats (10 - 12 weeks old) and Male Wistar Hannover rats (11 - 12 weeks old) used in the study were obtained from Japan SLC Inc. (Hamamatsu, Japan). A standard solid-meal commercial food (LabDiet®; Nippon Nousan Ltd., Yokohama, Japan) was used. All animal protocols were approved by the institutional animal care and use committee. Experiments were conducted in accordance with the Guidelines for Animal Experimentation, Kyoto Pharmaceutical University.
2.2. Preparation of Dissolving Microneedle Array Chips and Subcutaneous Solutions Containing OA
To produce the dense OA solution, hydrogel, 110, 140, or 170 μl of distilled water was added to the mixture of 2 mg, 10 mg, or 20 mg of OA, 0.1 mg of BB and 35 mg of sodium chondroitin sulfate and was kneaded at the low temperature, 15˚C. To produce two-layered microneedle array chips, the hydrogel was degassed under reduced pressure. It was then dispensed into a mold containing 225 inverted cone-shaped wells with an area of 2.25 cm2. Each well had 500 µm depth and 300 µm diameter at its top. The mold was covered with a 300 g steel plate; then the drug hydrogel was filled into the wells. After the plate was removed, hydrogel made of 15 mg of chondroitin sulfate and 25 ml of distilled water was painted over the mold. It was then dried under pressure of the stainless steel plate for 3 h. Thereafter, the plate was removed and the dissolving microneedle array chip was obtained by detaching them from a supporting material base. To produce a three-layered microneedle array chip, filling and drying were performed in the following order: 1) chondroitin sulfate hydrogel; 2) OA hydrogel; and 3) chondroitin sulfate hydrogel. To make FL-OA microneedle array chip, a mixture of OA and FL-OA (3:1) was used.
The OA solution for subcutaneous injection study was prepared by dissolving 10 mg, 100 mg, or 1000 mg of OA with 200 ml of phosphate buffered saline (PBS).
2.3. Microscopic Observation of Dissolving Microneedles
Dissolving microneedle array chips containing FL-OA were observed using a digital video microscope (VH- 5500; Keyence Co., Osaka, Japan) under normal light and under fluorescent light, 495 nm.
2.4. OA Content in Dissolving Microneedle Array Chip
The OA was extracted from dissolving microneedle array chip with 10.0 ml of 0.1 mM phosphate buffer, pH 7.4, and the OA contents were determined using an ELISA kit (Morinaga Institute of Biological Science, Inc., Tokyo, Japan).
2.5. Immunization Studies in Rats
Male Brown Norway rats, 10 - 12 weeks old at primary immunization, were anesthetized with intraperitoneal injection of sodium pentobarbital, 50 mg/kg. One group consisted of 4 - 5 rats. At 5 min before administration, 0.5 ml of blank blood samples was obtained from the left jugular vein using a heparinized syringe. Hair on the abdominal region was removed with a shaver (ES7111; Panasonic Inc., Osaka, Japan). The dissolving microneedles were inserted into the skin by pressing the chip with two fingers for 3 min with no treatment. The animals were boosted with the dissolving microneedles at 2 weeks after first immunization. Blood samples, 0.5 ml, were obtained from the left jugular vein using a heaprinized syringe at 2 weeks and 4 weeks after the first immunization. By centrifugation at 9000 ×g for 10 min at 4˚C using a centrifuge (Kubota 1700; Kubota Corp., Tokyo, Japan), 200 ml plasma samples was obtained. The resultant plasma samples were stored at −80˚C until analysis.
For the sc injection experiment, OA solution was injected to the abdominal region of rats at doses of 10, 100, and 1000 mg/rat. The animals were boosted with OA solution at 2 weeks after first immunization. At 2 and 4 weeks after first immunization, 0.5 ml of blood samples was obtained from the left jugular vein using a heparinnized syringe.
All these plasma samples were immediately frozen in a deep freezer at −80˚C until analysis.
2.6. Assay for Anti-OA Total Ig(G + A + M) and IgE Antibody
The OA-specific and IgE antibody concentrations in the rat plasma samples were determined using an ELISA kit (Alpha Diagnostic International, Inc., TX, USA). The concentration of anti-OA antibody of the plasma samples was determined against a standard curve of rat OA Ig calibrators. Plate washers were used (Dia-washer II; Dia Iatron Co. Ltd. USA). Absorbance was measured at 450 nm using a microplate reader (MTP-300; Corona Electric Co. Ltd., Japan). The rate of change of IgE level at 2 weeks after the start of transcutaneous immunization against the pretreatment level was calculated using the following equation.
2.7. In Vivo Delivery and Distribution Experiment of FL-OA in the Rat Skin
Male Wistar Hannover rats, 265 - 305 g, were used. Under anesthesia with an intraperitoneal injection of sodium pentobarbital, 50 mg/kg, the hair covering the abdominal skin was removed using an electric shaver. Dissolving microneedles containing FL-OA was administered to the skin. To study the delivery and diffusion rate of FL-OA to the vertical direction of the rat skin, skin samples were obtained at 30 s and at 2, 5, and 10 min after administration of the FL-OA dissolving microneedles to the rat skin. The skin samples were embedded (Histo Prep®; Fisher Scientific International, NJ, USA), frozen at −80˚C in a deep-freezer, and sectioned verticality using a microtome (THK; Kenis Ltd., Osaka, Japan). The skin sections, approximately 40 mm, were mounted on glass slides. The slides of FL-OA loaded rat skin were visualized—under normal light and the fluorescent light, 495 nm, with no staining or treatment—through a 10× objective using a video microscope (VH-5500; Keyence Co., Osaka, Japan) equipped with a filter for fluorescein observation.
To study the horizontal distribution of FL-OA in the rat skin, skin samples were obtained at 1 min after administration of the twoand three-layered dissolving microneedles containing FL-OA. The skin samples were embedded, frozen at −80˚C in a deep-freezer, and sectioned horizontally from the surface to the depth of 400 mm using a cryostat microtome (Leica CM 3050 S; Leica Inc., Nussloch, Germany). From each slice sample, 20 mm thick FL-OA was extracted using 700 ml of 0.1 N NaOH solution. After vortexing for 1 min and centrifugation at 9000 ×g for 5 min, 100 ml of supernate was used for measuring fluorescent intensity by Fluoroskan (Ascent CF; Labsystems, Helsinki, Finland), where excitation and emission wavelengths were, respectively, 485 and 538 nm. The distribution ratio of FL-OA in rat skin was calculated using the following equation.
2.8. Statistics
All data are shown as mean ± S.E. Statistical differences were inferred as significant when p < 0.05 or p < 0.01 (Student’s unpaired t-test).
3. Results
3.1. Physicochemical Properties of Dissolving Microneedles
The prepared twoand three-layered dissolving microneedles containing FL-OA are presented in Figure 1. In addition, the images of the enlarged dissolving microneedles are presented in Figure 2. As shown in those photos, FL-OA was formulated at the acral or second portions of dissolving microneedles. The physicochemical properties of the prepared dissolving microneedles are shown in Table 1. Mean lengths of the dissolving microneedles were, respectively, 480 ± 9 μm and 485 ± 7 μm for twoand three-layered dissolving microneedles. The basement diameters were, respectively, 275 ± 8 μm and 281 ± 3 μm. The mean length of the FL-OA loaded space was 0 - 155 ± 5 μm for two-layered and 175 ± 4 - 224 ± 5 μm for three-layered dissolving microneedles.
3.2. In Vivo Immunological Evaluation of Dissolving Microneedles in Rats
The obtained dissolving microneedles containing OA were administered to the rat skin by pressing the chips on the abdominal skin where the hair had been removed. After administration, the chips were recovered and the amounts of OA in the recovered chips were measured. Table 2 presents the OA contents in the chips and recovered amounts of OA in the chips.
In the case of two-layered OA microneedle array chips, the OA contents were, respectively, 2.2 ± 0.4, 12.6 ± 2.4, and 22.7 ± 1.3 mg. In contrast, the recovered OA in the chips were, respectively, 0.1 ± 0.1, 0.5 ± 0.2, and 0.7 ± 0.2 mg. By subtracting the recovered amount of OA from the amount of OA formulated in microneedle array chips, the administered doses of OA were estimated as 2.2 ± 0.1, 12.0 ± 0.2, and 22.0 ± 0.2 mg, respectively, as also shown
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.