Chitosan Composite Microneedles as a Single

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Sodium Hyaluronate/Chitosan Composite Microneedles as a Single-Dose Intradermal Immunization System Yu-Hsiu Chiu, Mei-Chin Chen, and Shu-Wen Wan Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b00441 • Publication Date (Web): 03 May 2018 Downloaded from http://pubs.acs.org on May 5, 2018

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Sodium Hyaluronate/Chitosan Composite Microneedles as a Single-Dose Intradermal Immunization System Yu-Hsiu Chiu1, Mei-Chin Chen1*, and Shu-Wen Wan2 1

Department of Chemical Engineering, National Cheng Kung University, Tainan, Taiwan

2

School of Medicine, College of Medicine, I-Shou University, Kaohsiung City, Taiwan

ABSTRACT: Enhancing the immune response to vaccines and minimizing the need for repeated inoculations remain a challenge in clinical vaccination. This study developed a composite microneedle (MN), composed of a sodium hyaluronate (HA) tip and a chitosan base, for biphasic antigen release and evaluated the potential of using this MN formulation as an intradermal delivery system for single-dose vaccination. Upon skin insertion, the dissolvable HA tip dissolved within the skin for rapid release of the encapsulated antigens, thus priming the immune system, while the biodegradable chitosan base remained in the dermis for prolonged antigen release for 4 weeks, thus further boosting the stimulated immunity. Our results showed that a single immunization with the HA/chitosan MN containing ovalbumin (OVA) (100 g  1) stimulated both T helper type 1 (Th1) and Th2 immune responses in rats and induced considerably higher and more durable antibody responses than a traditional two-dose (100 g OVA  2) or double-dose (200 g OVA  1) subcutaneous vaccination. Thus, the proposed MN exerts strong adjuvanticity to greatly augment the antigen’s immunogenicity. Moreover, given its unique rapid and sustained release properties, the HA/chitosan MN formulation has the potential

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to replace the conventional primeboost regimen to serve as an effective single-dose vaccine formulation.

KEYWORDS: adjuvanticity; biphasic release; cellular immunity; intradermal delivery; vaccination.

INTRODUCTION Traditional vaccines, especially inactivated and subunit vaccines, are often less immunogenic and require multiple booster doses to induce effective protection.1−3 Generally, the first injection activates the immune system but fails to elicit protective immunity.4,5 The second or third dose helps to generate high enough immunity to fully protect against pathogen infection. However, repeated injections increase the cost of immunization and are stressful and inconvenient to the vaccine recipient.1,3 Moreover, ensuring timely administration of a booster dose is problematic for people who have limited access to healthcare, which may result in ineffective immunization. Therefore, a single-dose vaccine is more practical and desirable than multiple dose regimens in improving the vaccine coverage rate. Using biodegradable polymers to develop controlled-release vaccine delivery systems has been recently considered a promising strategy for reducing the need for repeated vaccine injections.2,3,6−9 Injectable poly(lactic-co-glycolic acid) (PLGA) microparticles are the most widely investigated system for this purpose.6,10 These microparticles need be administered intramuscularly or subcutaneously only once, because they release encapsulated antigens in a sustained manner to induce both primary and secondary immune responses.3 However,


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maintaining antigen stability is a major challenge for such particulate vaccines because the labile antigens may become denatured and may lose their antigenicity due to contact with organic solvents during particle formulation.2,11 Chitosan, a natural and biodegradable polysaccharide, has been proposed as a potential alternative to PLGA because of its low cost, mild formulation procedures, and unique adjuvanticity.12 It can be processed in aqueous media without the need for chemical solvents or high temperature, allowing encapsulation of sensitive proteins and maintaining their bioactivity.13 Like PLGA microparticles, chitosan-based carriers can also act as a local depot at the inoculation site, resulting in a sustained release of antigens for a few weeks.14−16 In addition to prolonging antigen exposure, chitosan has been shown to possess the immunostimulatory activity that can promote dendritic cell activation and enhance antigen presentation, thus inducing cellular immunity.12,13 We previously reported that immunization using chitosan microneedles (MNs) can promote the immune response to encapsulated antigens, resulting in a noticeable dose reduction relative to intramuscular injection.14 Such enhancement is attributed to sustained intradermal (ID) delivery of antigens and to continuous activation of immune cells provided by chitosan MNs. This study focused on the potential of chitosan-based MNs as a single-administration delivery system for vaccines and explored the effect of antigen release behavior on elicited immune responses. In this study, we developed sodium hyaluronate (HA)/chitosan composite MNs, which allow both rapid and sustained release of antigens to mimic conventional prime-boost immunization regimens. We assume that the biphasic release profiles have the potential to stimulate superior immune responses compared with traditional bolus injection or continuous dosing with the chitosan MN alone.



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The composite MN was composed of a HA tip and a chitosan base, which was combined with a polyvinyl alcohol/polyvinyl pyrrolidone (PVA/PVP) supporting structure (Fig. 1). HA, also a naturally-occurring polysaccharide, was used as a tip material because of its biocompatibility, proven safety record, and highly hydrophilic nature.17,18 Upon skin insertion, the supporting structure offered additional length for complete MN insertion and was dissolved in the skin within a few minutes (Fig. 1a). The antigen-loaded HA/chitosan MNs can be subsequently implanted into the dermis for two-stage ID vaccination (Fig. 1b). First, the antigen is rapidly released from the dissolving HA tip to prime the immune system. After the tip has dissolved, the chitosan base provides a sustained release of antigens to further boost the stimulated immune response. We hypothesize that the rapid and sustained antigen release from the proposed MN enables the replacement of multiple-injection vaccination regimens and evokes rapid and effective responses to the antigen. To investigate the potential of the HA/chitosan MNs as a single-dose vaccine formulation, we compared the antibody levels generated by a single MN immunization (single dose) with those generated by the conventional prime-boost regimen (two doses). To further explore the effect of antigen release kinetics on their elicited immune responses, the type and magnitude of the immune responses in rats immunized with the composite MNs were compared with those of rats immunized with the chitosan MNs or subcutaneous (SC) injections. Moreover, the skin insertion ability, antigen release behavior, and in vivo MN degradability were evaluated.


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Figure 1. Schematic illustrations of composite microneedles (MNs), composed of a sodium hyaluronate (HA) tip and a chitosan (CS) base, with dissolvable supporting structures for biphasic antigen release. Upon skin insertion, the supporting structure offered additional length for complete MN insertion and was dissolved in the skin within 4 min (a). The antigen-loaded MNs can be subsequently implanted into the dermis for rapid release of antigens from the HA tip to prime the immune system and sustained release of antigens from the CS base to boost the stimulated immunity (b).



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EXPERIMENTAL SECTION Materials: HA with molecular weight (MW) of 7 and 200500 kDa was purchased from Bloomage Freda Biopharm (Jinan, China) and Kewpie (Tokyo, Japan), respectively. Chitosan (viscosity = 22 mPa·s for 3.2% in 1% acetic acid at 20 oC, and degree of deacetylation = 91.2%,), Poly(L-lactide-co-D,L-lactide) (PLA; L-lactide:D,L-lactide = 70:30, inherent viscosity = 1.82.4 dL/g), and PVA (MW = 6000) were obtained from Koyo Chemical (Osaka, Japan), Green Square Materials (Taoyuan, Taiwan), and Polysciences (Warrington, PA, USA). Rhodamine 6G (MW = 470), fluorescein 5(6)-isothiocyanate (FITC; MW = 389), blue dextran (MW = 2000 kDa), PVP (MW = 10000), and ovalbumin (OVA; from chicken egg white, MW = 44.3 kDa) were purchased from Sigma－Aldrich (St. Louis, MO, USA).

Fabrication of HA/Chitosan MNs with PVA/PVP Supporting Structures: Stainless steel master structures of MNs and pressing tools were obtained from Hong Da Precise Industry (New Taipei City, Taiwan). Negative polydimethylsiloxane (PDMS) molds were fabricated by replicating these master structures according to a published method.19 An OVA-containing HA solution was prepared by dissolving HA powder (weight ratio of 7 kDa:200500 kDa = 1:1) in OVA aqueous solution to form a 3 wt% HA solution. To obtain the OVA-containing chitosan gel, OVA was first dissolved in a trehalose aqueous solution and then blended with chitosan gel (5 wt%).19 The mixture was evaporated at 37 C to form a viscous gel containing OVA, 1.8 wt% trehalose, and 18 wt% chitosan for casting. The composite HA/CS MNs were fabricated using a multilayer casting method (Fig. 2). To form the OVA-loaded HA tip, the OVA/HA solution (200 μL) was placed on the PDMS mold and


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centrifuged in a swinging bucket rotor at 5000 rpm at 25 °C for 10 min. The excess solution was collected for later use. The filled mold was centrifuged again at the same speed and temperature for 2 h without sealing caps of centrifuge tubes for drying.

Figure 2. Schematic illustrations of the fabrication process for the HA/Chitosan MNs with polyvinyl alcohol/polyvinyl pyrrolidone (PVA/PVP) supporting structures. OVA: ovalbumin; PLA: poly(L-lactide-co-D,L-lactide). To make the chitosan base, the OVA/trehalose/chitosan gel (200 mg) was added onto the mold, which was centrifuged under the same conditions for 2 h. The gel remaining on the mold’s surface was collected for later use. After drying at room temperature (RT) for 10 min, a PLA pressing tool was used to push the gel into the mold cavities. The chitosan-gel filling process was repeated two times to produce a solid MN.



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To prepare the supporting structure, a 50 wt% PVA/PVP aqueous solution (0.5 g, weight ratio of PVA:PVP = 1:1) was added on the mold and then placed in a vacuum oven (−40 cm Hg) at RT for 10 min. The solution remaining on the mold surface was scraped off to remove the bubbles formed during vacuuming. The 50 wt% PVA/PVP solution (0.5 g) was added again to form a patch. After air-drying for 1 day, the filled mold was placed in an oven at 37 °C for 1 day. The process for fabricating the PLA pressing tool is described in Supporting Information. To visualize the composite MNs in the skin, HA and chitosan were fluorescently labeled with rhodamine 6G (R6GHA)20 and FITC (FITC–chitosan)21, respectively. Quantification of Antigen Loading: To measure the OVA amount in MNs, all needles were cut off from the patch with a razor blade and soaked in a 0.1% (v/v) acetic acid solution under stirring for 5 days (100 rpm, 4 °C) until all samples were dissolved. The amount of antigen extracted from the needles was determined using a Pierce BCA Protein Assay Kit. Ethics Statement: All experimental animal procedures were ratified by the Institutional Animal Care and Use Committee of National Cheng Kung University (NCKU), and all the experiments were implemented according to the guidelines of the Laboratory Animal Center of NCKU. In Vitro and in Vivo Skin Insertion: The OVA-loaded MNs were inserted into porcine cadaver skin or rat dorsal skin by using a homemade applicator (approximately 10 N/patch) for 4 min. Before the MN was applied, the dorsal hair of each Sprague–Dawley rat (male, 4–5 weeks old) was carefully shaved. The rats were sacrificed at specific time intervals and the MN-treated skin was excised for confocal microscopic and histological analysis. Three-dimensional (3D) confocal reconstruction images were obtained through xyz stack acquisition to visualize OVA release in the skin.


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Franz Diffusion Cell Method: In vitro antigen transdermal delivery was conducted using a Franz diffusion cell apparatus with a permeation area of approximately 0.66 cm2. First, the SC fat of the porcine cadaver skin was removed and OVA-loaded MNs were inserted into the skin. To compare the antigen release from the different MN matrices, Texas Red OVA conjugate (Texas RedOVA) and FITC-labeled OVA (FITCOVA) were encapsulated in the HA tip and chitosan base, respectively. The MN-treated skin was placed on the cell by providing dermis contact with the receptor compartment filled with 5 mL phosphate buffered saline (PBS, pH 6.2). The receptor solution was stirred at 220 rpm and maintained at 32 ± 1 °C by using a circulating water jacket. At selected time intervals, the receptor solution was removed for concentration measurement and replaced with an equal volume of fresh PBS. The OVA amount in the receptor solution was determined by measuring the fluorescence intensity of fluorescently labeled OVA from the supernatant. In Vivo Fluorescence Imaging: To observe antigen retention in the skin, live whole animal imaging was conducted on anesthetized rats by using an in vivo imaging system (IVIS). Fluorescence data were processed using a region of interest analysis with background subtraction and normalized to the fluorescence value at Day 1 after MN treatment.22,23 Immunization in Sprague–Dawley Rats: Rats (male, 4–5 weeks of age) were randomly assigned into seven groups (n = 6 rats for each group): (1) an unimmunized control group, in which animals received SC injection of saline (100 μL); (2) an SC single-dose group (100 g OVA  1), in which animals were immunized by SC injection with a single dose of OVA (100 g in 50 L PBS); (3) an SC double-dose group (200 g OVA  1), in which animals were immunized by SC injection with a double dose of OVA (200 g in 100 L PBS); (4) an SC two-dose group (100



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g OVA  2), in which animals were SC inoculated with OVA at Day 0 and boosted 2 weeks later; (5) an SC single-dose plus polymer group (100 g OVA  1), in which rats were SC inoculated with a single dose of OVA and the same amounts of HA (0.5 mg), trehalose (0.13 mg), and chitosan (1.3 mg) solution as in group (7); and (6) a chitosan MN (100 g OVA/patch  1) group and (7) composite HA/chitosan MN group (100 g OVA/patch  1: 30 g OVA in HA/70 g OVA in chitosan), in which animals received single-dose MN immunization. Blood was sampled at predetermined time intervals and permitted to clot at 4 °C for 1 h. Serum was separated by centrifugation and then stored at −20 °C until use. Quantitation of Anti-OVA Antibodies: OVA-specific antibodies were measured using enzymelinked immunosorbent assay. Each well on 96-well plates was first coated with OVA (2 g/well) diluted in carbonate buffer (0.05 M, pH 9.6) at RT for 1.5 h and then blocked with 1% bovine serum albumin (BSA) in PBS at RT for 1.5 h. Wells were washed three times with PBS containing 0.05% Tween 20. Sera were diluted to 1:1000 with PBS containing 0.66% BSA for measuring the total immunoglobulin G (IgG) levels or diluted serially for determining the IgG1 and IgG2a titers. Next, the coated wells were incubated with dilutions (100 μL/well) of serum at RT for 1.5 h. After washing, the wells were incubated with horseradish peroxidase-conjugated goat antibody specific for rat IgG, IgG1, and IgG2a (1:10,000; 100 μL/well). The plates were rewashed to remove unbound antibodies and were developed using 2,2'-azino-bis(3ethylbenzothiazoline-6-sulphonic acid) substrate (100 μL/well). The color was allowed to develop until the OD405 of the highest absorbing well reached approximately 1.5. Titers were expressed as the reciprocal of the highest dilution providing an absorbance of OD405 > 0.12 above the sera of the unimmunized control group.


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Statistical Analysis: The differences between two groups were analyzed using the one-tailed Student’s t test by using SPSS (Chicago, Ill, USA). Data are presented as mean ± standard deviation (SD). A p value of < 0.05 was considered to indicate a statistically significant difference. RESULTS AND DISCUSSION Characterization of HA/Chitosan MNs with Supporting Structures: Figure 3a and 3b show the bright-field images of the array of the composite HA/chitosan MNs with PVA/PVP supporting structures. To distinguish the HA tip from the chitosan base, HA was blended with a dye marker, blue dextran, and chitosan was labeled with a fluorescence dye, FITC. As shown in Figure 3a and 3b, we successfully developed a pyramidal-shaped MN composed of an HA tip (blue) and chitosan base (yellow). The prepared MN device consisted of 81 needles in a 9 × 9 pattern. Both the MN and supporting structure had a length of 550 μm and a base width of 300 μm. The inset in Figure 3 provides the specifications of the fabricated MN device. Skin Insertion Ability: The fabricated MN system was applied to the porcine cadaver skin (Fig. 3c) and rat skin (Fig. 3d3f) to evaluate its skin puncture ability. After MN insertion, we observed an array (9  9) of microholes on the skin surface (Fig. 3c and 3d), confirming that the composite MNs have enough mechanical strength to achieve stable and repeatable puncture results. Figure 3e and 3f present histological sections of the rat skin after application of R6GHA/FITCchitosan MNs. As shown, the composite MNs can pierce through stratum corneum and then be implanted in the dermal layer, with an insertion depth of 612 ± 46 m (n = 5). The thicknesses of the epidermal and dermal layers of the tested rats was approximately 60 and 1000 m (n = 5), respectively.14



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The red fluorescence signal (R6GHA) surrounded the green one (FITCchitosan), indicating that the interstitial fluid can easily dissolve the HA tips, leaving only the chitosan base in the skin (Fig. 3f). We assume that the dissolving HA tip allows for rapid release of the encapsulated antigens to rapidly activate the immune system, while the degradable chitosan base enables a sustained delivery of antigens to continually amplify the induced immunity.

Figure 3. Bright-field micrographs of the composite MNs, composed of an HA tip (blue) and a chitosan base (yellow), with PVA/PVP supporting structures (a and b). To distinguish the HA tip from the chitosan base, HA was blended with a dye marker, blue dextran, and chitosan was labeled with a fluorescence dye, FITC. Bright-field micrographs of a porcine cadaver skin (c) and a rat skin (d) after application of R6GHA/FITCchitosan MNs. Histological sections of the MN-treated rat skin: bright-field (e) and fluorescence (f) images. The red fluorescence in (f)


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indicates the R6GHA; the green fluorescence in (f) indicates the FITC-chitosan MNs. The inset displays the detailed dimensions of the composite MN. In Vitro Antigen Release Profiles: The OVA was labeled with different fluorophores and separately encapsulated in the HA tip and chitosan base of the MN to monitor the antigen release from these two matrices. These OVA-loaded HA/chitosan MNs were applied to porcine skin first, and the antigen permeation across the cadaver skin was studied using a Franz diffusion cell. We found that almost all OVA loaded in the HA tips was released and penetrated across the skin within 7 days (Fig. 4). Compared with the HA tip, only approximately 35% OVA was released from the chitosan base during this period. These results showed that using hydrophilic HA as the tip material results in a rapid release of OVA, whereas slow and prolonged release of antigen can be achieved by encapsulating OVA in the chitosan matrix.

Figure 4. In vitro OVA release from the HA tip and the chitosan (CS) base, conducted using Franz diffusion cells (n  4 MN patches). Data are presented as mean ± SD.



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In Vivo Antigen Release and MN Degradability: In vivo fluorescence images were acquired using IVIS to determine the retention time of OVA in rat skin following MN delivery (Fig. 5). To evaluate the effect of the polymer properties on the antigen release, FITCOVA and Texas RedOVA were loaded in the HA tip and chitosan base, respectively. Figure 5b and 5c show that the intensity of the FITCOVA signal quickly declined and became undetectable within 1 week, indicating that OVA released from the HA tips disappeared from the insertion site. Because of the hydrophilic nature of HA, the HA tip can be easily dissolved in the skin (Fig. 3f) and may form a viscous HA gel at the insertion site. The OVA release from the HA matrix is mainly determined by the HA dissolution and the antigen diffusion through the viscous HA gel.24,25


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Figure 5. In vivo OVA retention within the skin. Fluorescence images of the whole rat body (a) and the MN-treated sites at specific time points (b) after insertion of the OVA-loaded HA/chitosan (CS) MNs. The percentage of OVA remaining in the rat skin was obtained by measuring a region of interest analysis with background subtraction and normalized to the fluorescence value at Day 1 after MN treatment (c) (n = 4 rats for each time point). Here, FITCOVA (green) and Texas RedOVA (red) were loaded in the HA tip and chitosan base, respectively. Data in (c) are presented as mean ± SD. The fluorescence intensity of the Texas RedOVA decreased slowly and was still detected after 28 days, demonstrating that the chitosan base can retain the antigen at the delivery site for prolonged antigen exposure of up to 4 weeks (Fig. 5b and 5c). Upon insertion, initial antigen release from the chitosan base may be due to the hydration and swelling of the chitosan matrix when contact with the skin interstitial fluid. After the swelling equilibrium was reached, OVA could be continuously released through slow diffusion out of and by degradation of the chitosan base.19,26 To visualize the spatial distribution of delivered OVA in the skin, we used confocal microscopy to generate 3D images of the rat skin after MN insertion (Fig. 6). Here, the Alexa Fluor 647 OVA conjugate (Alexa 647OVA) and Texas RedOVA were separately encapsulated in the HA tip and FITCchitosan base. Figure 6 shows the reconstructed 3D confocal micrographs of the skin taken on different days. As shown, the blue fluorescence of the Alexa 647OVA was only visible on Day 0 and undetectable on Day 7, indicating that the OVA was quickly released from the HA tip within 7 days. Conversely, the OVA (red) encapsulated in the chitosan base (green) was clearly observed in the skin until 28 days, revealing that the chitosan base can function as a depot for slow and targeted release of antigen into the skin. Additionally, we found



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that the red fluorescence intensity of the Texas RedOVA gradually decreased with the reduced green signal, suggesting that the antigen retention and release was highly correlated with the chitosan degradation.

Figure 6. In vivo OVA release from the implanted MNs. The reconstructed 3D confocal micrographs of the MN-treated rat skin. Here, Alexa 647OVA (blue) and Texas RedOVA (red) were separately encapsulated in the HA tip and FITCchitosan base (green). To observe the degradation of the chitosan base in the skin, the MN-treated rat skins were processed for histological examination (Fig. 7). We found the implanted chitosan base (green) gradually became small and finally disappeared from the skin after 28 days. These results were consistent with the results obtained by confocal microscopy (green in Fig. 6) and demonstrated that the chitosan base can be completely degraded in the skin within 1 month.14,19 The implanted


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base displays yellow fluorescence in the merged images, suggesting that the OVA (red) was uniformly distributed in the chitosan base (green). Moreover, very weak red fluorescence was found around the chitosan base (merged image), indicating that the encapsulated OVA was slowly released from the base. Even after 28 days, fragments of the chitosan base still contained OVA, further demonstrating its antigen depot function.

Figure 7. In vivo degradation of the chitosan base. Histological sections of the MN-treated rat skin. Here, Texas RedOVA (red) was loaded in the FITCchitosan (green) base. Potential of Using HA/Chitosan MNs for Single-Dose Vaccination: Our previous study confirmed that chitosan MNs can provide sustained antigen release and immunostimulating activity to considerably augment the antigen-specific antibody response.14 However, that study focused on the effect of the chitosan MN’s adjuvanticity, and it remains unclear whether adjusting the antigen release kinetics would further promote the durability and strength of immune responses. In the present study, we integrated a dissolving HA matrix into the chitosan



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MN system to produce a rapid and extended release formulation for intradermal vaccination. We hypothesized that such a biphasic antigen release would better mimic a conventional vaccination regimen involving prime and booster doses and thus has potential to reduce the requirement for repeated immunizations. To evaluate the potency of the HA/chitosan MNs as a single-dose formulation for vaccines and whether the proposed MNs with rapid and sustained release properties would stimulate superior immunity than the chitosan MNs, Sprague–Dawley rats were immunized with the HA/chitosan MNs containing OVA (100 g  1) and compared with rats immunized with SC injections of single-dose (100 g  1), double-dose (200 g  1), two-dose (100 g  2, i.e., the prime-boost regimen) OVA or single-dose OVA (100 g  1) plus HA and chitosan solution, and chitosan MNs containing OVA (100 g  1). Figure 8 shows (a) the OVA-specific IgG levels and (b) the ratio of IgG1 to IgG2a titers after immunization.

Figure 8. Immune responses in rats after MN immunization (n = 6 rats for each group): OVAspecific IgG levels (a) and the ratio of IgG1/IgG2a titers (b) in serum. Animals were immunized by subcutaneous (SC) injection with a single dose of OVA (SC single-dose, 100 g OVA  1), a double dose of OVA (SC double-dose, 200 g OVA  1), or a single dose of OVA plus the same


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amounts of HA and chitosan (SC single-dose plus polymer, 100 g OVA  1) as in the HA/chitosan MN group; animals were SC inoculated with OVA at Day 0 and boosted 2 weeks later (SC two-dose, 100 g OVA  2); animals received single-dose MN immunization (100 g OVA  1): chitosan MN and HA/chitosan MN. Each bar in (a) represents mean ± SD. *: p < 0.05; **: p < 0.01; ***: p < 0.001. The actual OVA amounts in the HA tip and the chitosan base were 30.0  2.2 and 70.7  1.1 g per composite MN patch (n  4 patches), respectively. As shown in Figure 8a, the SC two-dose group (100 g  2) resulted in significantly higher IgG levels than the SC single-dose group (100 g  1) from week 4 after immunization (p  0.01, n  6), showing a booster dose indeed stimulated more antibody production and induced longerlasting antibody level than one standard dose did. Additionally, animals received OVA-loaded chitosan MNs (100 g  1) elicited robust anti-OVA IgG antibody levels, which were either higher than or equal to the levels detected in the SC two-dose (100 g  2) or SC double-dose (200 g  1) group. These results reveal that use of chitosan-based MNs for immunization can enhance antigen immunogenicity, thus providing a remarkable dose-sparing benefit to reduce vaccination costs. Most importantly, we observed that the HA/chitosan MNs can quickly induce antibody responses at week 2 after immunization and result in significant increases in specific IgG serum antibody levels compared with the chitosan MN group (Fig. 8a, p  0.001, n  6). These immune responses can be maintained at relatively high levels for at least 16 weeks through single administration. Our results indicate that not only the adjuvant but also the antigen release behavior could play a pivotal role in influencing the development of antigen-specific immune responses. Although both the composite MNs and the chitosan MNs contain the same dose of



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OVA (100 g  1), immunization through the HA/chitosan MNs can induce significantly faster and greater immunity compared with the chitosan MNs (p  0.001, n  6), demonstrating that the biphasic release system (combination of rapid and sustained release) would be more beneficial for stimulating antibody production than a conventional sustained delivery one. When comparing the same dose of OVA and polymers, the HA/chitosan MN group elicited considerably stronger antibody responses than did the SC single-dose plus polymer group (p  0.001, n  6), showing that encapsulation and delivery of antigen using the proposed MNs can further improve the antigen’s immunogenicity. Additionally, the polymer’s adjuvanticity can be further enhanced in the MN form than in the solution form. These improvements may be attributed to the unique antigen retention and biphasic release properties of the HA/chitosan MNs. Next, we analyzed the IgG subclass levels and assessed whether the immune response elicited by the new MN formulation was polarized towards T helper type 1 (Th1) or type 2 (Th2) immunity. Generally, it is accepted that IgG1 is a marker for Th2-type humoral immunity, whereas IgG2a is associated with a Th1-skewed cellular response.27,28 We found that SC vaccination with OVA alone tended to stimulate IgG1 secretion, resulting in an IgG1/IgG2a ratio 1 (Fig. 8b). This suggests that the OVA itself preferentially induced Th2-type immunity. By contrast, immunization with the OVA-loaded chitosan or HA/chitosan MNs or OVA plus HA and chitosan solution enhanced both IgG1 and IgG2a responses, with a more balanced IgG1/IgG2a ratio. Additionally, the IgG1/IgG2a ratio of these two MN groups did not differ substantially. These results indicate that the chitosan-based formulation is capable of directing the immune response from mainly Th2 to a more balanced Th1/Th2 response. A recent study highlighted the ability of chitosan to activate dendritic cells and promote cellular immunity.12


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Biomacromolecules

HA is the most commonly used materials for dissolving MNs and these HA-based MNs were able to rapidly deliver the encapsulated antigen and effectively induce robust antibody responses that were comparable or superior to those generated by conventional needle injections.2932 However, these dissolving HA MNs can only serve as convenient and reliable alternatives to needle-based immunization, but still need repeated administrations to achieve a long-lasting immunity. In this study, we demonstrate the HA/chitosan MNs has potential as a single-dose antigen delivery system to minimize the need for repeated inoculations. The proposed MN system can provide a biphasic release pattern to mimic a multiple-dose vaccine schedule. After single immunization in rats, the HA/chitosan MNs induced greater and longer-lasting antibody responses than did repeated (100 g  2) or double-dose (200 g  1) SC vaccination (Fig. 8a). Additionally, compared to the chitosan MN system, the composite MNs showed stronger enhancement in the antigen’s immunogenicity. These results demonstrated that the combination of a rapid and sustained release is more favorable than a bolus or a sustained release pattern to augment immune responses.

CONCLUSIONS This study emphasizes the potential of HA/chitosan MNs as an efficient ID antigen delivery tool for single-dose vaccination. We demonstrated that single-dose immunization with the composite MNs generated robust and persistent IgG antibody levels for at least 16 weeks, substantially higher than the levels induced by prime-boost SC or chitosan MN-based vaccination. Thus, the unique adjuvanticity and biphasic release properties of the HA/chitosan MN system greatly enhances the immunogenicity of delivered antigens. Accordingly, the proposed MN delivery



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technology may increase immunization coverage by reducing the number of inoculations or the vaccine dose required for effective vaccination.

ASSOCIATED CONTENT Supporting Information: fabrication method of the PLA pressing tool.

AUTHOR INFORMATION Corresponding Author: E-mail: *[email protected] ACKNOWLEDGMENTS: We acknowledge the assistance from the laboratory animal center of NCKU and the technical services provided by the Bio-image Core Facility of the National Core Facility Program for Biotechnology, Ministry of Science and Technology, Taiwan. We also gratefully appreciate the financial support of the Ministry of Science and Technology of Taiwan (MOST 105-2628-B-006-008-MY3 and MOST 106-2221-E-006-058-MY3) and Wallace Academic Editing for editing this manuscript.

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For Table of Contents Use Only

Sodium Hyaluronate/Chitosan Composite Microneedles as a Single-Dose Intradermal Immunization System Yu-Hsiu Chiu1, Mei-Chin Chen1*, and Shu-Wen Wan2


Chitosan Composite Microneedles as a Single

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