Journal of Controlled Release 196 (2014) 252–260

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Microprojection arrays to immunise at mucosal surfaces Celia L. McNeilly a,1,2, Michael L. Crichton a,b,1, Clare A. Primiero a, Ian H. Frazer c, Michael S. Roberts d,e, Mark A.F. Kendall a,b,c,⁎ a

The University of Queensland, Delivery of Drugs and Genes Group (D2G2), The Australian Institute for Bioengineering and Nanotechnology, St Lucia, QLD 4072, Australia Vaxxas Pty Ltd, Australian Institute for Bioengineering and Nanotechnology, Brisbane, Queensland, Australia c The University of Queensland Diamantina Institute, Translational Research Institute, Brisbane, Queensland, Australia d Therapeutics Research Centre, School of Medicine, Princess Alexandra Hospital, The University of Queensland, Brisbane, Queensland, Australia e School of Pharmacy and Medical Science, University of South Australia, Adelaide, Australia b

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Article history: Received 18 June 2014 Accepted 29 September 2014 Available online 5 October 2014 Keywords: Mucosa Immune response Drug delivery Mechanical properties

a b s t r a c t The buccal mucosa (inner cheek) is an attractive site for delivery of immunotherapeutics, due to its ease of access and rich antigen presenting cell (APC) distribution. However, to date, most delivery methods to the buccal mucosa have only been topical—with the challenges of: 1) an environment where significant biomolecule degradation may occur; 2) inability to reach the APCs that are located deep in the epithelium and lamina propria; and 3) salivary flow and mucous secretion that may result in removal of the therapeutic agent before absorption has taken place. To overcome these challenges and achieve consistent, repeatable targeted delivery of immunotherapeutics to within the buccal mucosa (not merely on to the surface), we utilised microprojection arrays (Nanopatches—110 μm length projections, 3364 projections, 16 mm2 surface area) with a purpose built clip applicator. The mechanical application of Nanopatches bearing a dry-coated vaccine (commercial influenza vaccine, as a test case immunotherapeutic) released the vaccine to a depth of 47.8 ± 14.8 μm (mean ± SD, n = 4), in the mouse buccal mucosa (measured using fluorescent delivered dyes and CryoSEM). This location is in the direct vicinity of APCs, facilitating antigenic uptake. Resultant systemic immune responses were similar to systemic immunization methods, and superior to comparative orally immunised mice. This confirms the Nanopatch administered vaccine was delivered into the buccal mucosa and not ingested. This study demonstrates a minimally-invasive delivery device with rapid (2 min of application time), accurate and consistent release of immunotherapeutics in to the buccal mucosa—that conceptually can be extended in to human use for broad and practical utility. © 2014 Elsevier B.V. All rights reserved.

1. Introduction An improved reach of effective vaccines to more people globally is a priority. With most of today's vaccines being administered by the needle and syringe (in liquid form), one significant way of improving vaccines is by moving to alternative (needle-free) delivery methods which vaccinate at sites rich in immune cells (e.g., the skin or mucosal sites). This contrasts with the current conventional immunisation strategy of intra-muscular injection which delivers to a tissue low in antigen presenting cells (APCs), critical cells required for the initiation of adaptive immune responses. Novel delivery site and method advantages include: avoiding needle-phobia [1]; eliminating unsafe needle practices ⁎ Correspondinga author at: The Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Corner Cooper and College Rd, St Lucia, QLD 4072, Australia. Tel.: +61 7 3346 4203; fax: +61 7 3346 3973. E-mail address: [email protected] (M.A.F. Kendall). 1 C.L. McNeilly and M.L. Crichton contributed equally to this work. 2 Current affiliation: Bacterial Pathogenesis Laboratory, QIMR Berghofer Medical Research Institute, 300 Herston Rd, Herston, QLD 4006, Australia.

http://dx.doi.org/10.1016/j.jconrel.2014.09.028 0168-3659/© 2014 Elsevier B.V. All rights reserved.

[2]; improving vaccine stability (indeed potentially removing the ‘cold chain’ with vaccines stored in dry form) [3]; dose reduction and improved immune responses [4,5]. In order to realise these potential benefits, the initial site of infection and the nature of the pathogen against which a protective immune response is desired must be taken into consideration. For many pathogens, this initial site is at a mucosal surface that is exposed to the environment and requires the rapid secretion of mucosal immunoglobulins to prevent the dissemination of infection. The ability of an antigen to induce mucosal as well as systemic immune responses depends on the cells targeted at the initial site of immunisation (reviewed in [6,7]). However, systemic routes of immunisation alone may not generate immune responses that are capable of trafficking specific antibodies to these mucosal sites. In a recent study by O'Meara et al. [8], a chlamydial vaccine antigen in combination with adjuvant delivered intra-nasally protected mice from infection but not from disease pathology, whereas the same antigen-adjuvant combination delivered trans-cutaneously protected mice from disease pathology yet had no effect on reducing the burden of infection. This study highlights the need for continued

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development of mucosal and systemic vaccine delivery systems for the generation of optimal immune responses. Additional benefits of new mucosal vaccine delivery systems (or indeed, epithelial vaccines) include protection of the antigen from physical elimination or degradation by enzymes, direct deposition and release of the antigen to, or near APCs within the mucosa and stimulation of the immune system to generate rapid adaptive and long lived protective immunity. Recently, the feasibility of delivering vaccines to the oral mucosa using microneedles has been demonstrated, with Ma et al. [9] showing that vaccine delivery into the lip or dorsal tongue surface of rabbits was able to induce both mucosal and systemic immune responses. A mucosal site that has been largely overlooked in developing new mucosal vaccine delivery technologies is the buccal mucosa; the inside lining of the cheek that forms part of the oral mucosa. If consistently and repeatedly targeted, the buccal mucosa offers a site rich in APCs [10] that is also a convenient, accessible part of the mucosa. So far, mechanical targeting methods have deposited many drugs—but few vaccines—on to the buccal mucosa surface [11,12]. As a device, there may be significant mechanical benefits to delivering to the buccal mucosa—several studies have identified significant complexity in breaching skin by mechanical means, with consistency of targeting [13–17]. Whilst the buccal mucosa layers are significantly thicker than skin's epithelia, its benefits include: 1. A much simpler surface layer to breach, due to the lack of a very tough keratinised layer. Although some animals including mice have a thin mucosal keratinised layer, it is still thinner than the corresponding skin surface. 2. The buccal mucosa surface is highly and consistently hydrated. In contrast, the skin surface can have a highly variable hydration—leading to variable skin mechanical properties [18,19]. Goktas et al. [20] found mucosal ultimate tensile stress to be ~1 MPa; an order of magnitude lower than skin. 3. It lacks the hairs, sun damage, scars and cosmetic effects that cause the skin to be a highly variable surface. However, there are other potential sources of buccal variability including, for example in humans, the buccal mucosa is covered with a salivary layer (0.1–0.07 mm thick [21] with a ~0.5 ml/min flow rate [22]) that has the potential to dilute or wash away vaccines placed on the surface. In this paper, we present a new minimally-invasive mechanical method that directly delivers drugs into the buccal mucosa (using vaccines as a test-case), by breaching the surface layer—as a practical way to achieve rapid, consistent and repeatable drug delivery. We utilised the Nanopatch, a small array of densely packed (N 20,000 cm− 2) micro-projections that has previously been shown to be effective in delivering vaccines to APCs in skin (there are many investigations targeting vaccines to skin; with [4,5,23–30] just being a subset). We conceived, developed and investigated the application of the Nanopatch to the mucosa. Dynamic application of the Nanopatch to the buccal mucosa using a simple clip applicator provides a practical means for rapid, controlled and consistent mucosal delivery of immunotherapeutics. We selected the mouse model and an influenza vaccine as the test-case immunotherapeutic vaccine. We show here that our approach delivers vaccine antigen to the interstitial space within the epithelium where APCs reside, and results in immune responses comparable to needlebased intramuscular injection, Nanopatch skin delivery and superior to oral vaccine delivery.

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approval of the Office of Gene Technology and Recombination (Australia). MacGreen mice carry the enhanced green fluorescence protein (eGFP) under the cfs1r promoter. In these animals all macrophage and monocyte derived cells express eGFP, including dendritic cells and Langerhans cells.

2.2. Clip applicator design Application of Nanopatches to the buccal mucosa was performed using a clip applicator methodology—shown schematically in Fig. 1. The clip utilised was a hinged clip with a spring that resulted in a closing force of 1.9 Newtons (+/−8%). The diameter of the clip end was ~5 mm to ensure that it would fit within the small target area of a mouse mouth. The patch was attached to one arm of the clip applicator using double sided tape.

2.3. Nanopatches and coating Nanopatches were manufactured by Deep Reactive Iron Etching as previously described [25] in the Rutherford Appleton Laboratory (Oxford, UK). The Nanopatches are etched from silicon and sputter-coated with a thin layer of gold. Each Nanopatch is 16 mm2 (4 mm × 4 mm) and contains 3364 projections of 110 μm length and spaced at 70 μm intervals. Nanopatches were cleaned in a solution of glycerol and water (1:1 ratio), flushed with an excess of water and allowed to dry prior to coating. Formulations for coating contained 1% methylcellulose (Sigma-Aldrich, Castle Hill, NSW, Australia), active agent (0.2 μm, yellow–green fluorescent FluoSpheres® (0.4%), DiD (20 μM) (both Invitrogen Australia Pty Ltd, Mulgrave, VIC, Australia) or the purified, inactivated detergent-disrupted split virion influenza vaccine, Fluvax 2010, that contains 30 μg/ml hemagglutinin of each of A/California/7/ 2009, A/Wisconsin/15/2009 (A/Perth/16/2009 like) and B/Brisbane/60/ 2008 (CSL Ltd, Parkville, VIC, Australia)) and PBS if required. 8 μl of coating solution was pipetted onto each Nanopatch and dried using a nitrogen gas jet coating procedure as previously described [25].

2. Materials and methods 2.1. Animals Adult female C57Bl/6 mice and MacGreen transgenic mice aged 6–12 weeks were used in this study according to the University of Queensland animal ethics regulations (AIBN/020/10) and with the

Fig. 1. The concept of applying the Nanopatch to the buccal mucosa of a mouse using a clip applicator to target APCs in the epithelium and lamina propria of the mucosa. This method uniquely places vaccine within the mucosa using a pain-free method.

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2.4. Nanopatch application to skin and mucosa Prior to Nanopatch application animals were anaesthetised with an intra-peritoneal injection of a Ketamine (80 mg/kg)/Xylasil (10 mg/kg) mixture (both Troy Laboratories Pty Ltd, Glendenning, NSW, Australia). Subsequently, the Nanopatch containing end of the clip applicator was placed inside the mouth of the animal (which was held open using tweezers) and the clip was then released to firmly hold the patch on the buccal mucosa. This was maintained there for 2 min prior to removal. To determine the forces that the clip applied to the surface of the mucosa a PCB load cell transducer (PCB Electronics Inc., New York, USA) was used. This was attached to a National Instruments 6221 DAQ unit which was operated through a LabVIEW (Nation Instruments, Texas, USA) programme to observe forces. By pushing the clip onto the load cell and using a moment arm balance to determine the clip opening force, the application force was determined. 2.5. Quantification of antigen delivered using a radioactive labelled tracer protein To quantify the amount of Fluvax2010® vaccine delivered to the buccal mucosa by Nanopatch application, C14-radioactive isotype labelled ovalbumin protein (25 nCi per Nanopatch) (American Radiolabeled Chemicals, St Louis, MO, USA) was used as a tracer in the Fluvax2010® coating formulation using a modified protocol previously described for skin [13]. This quantification relies on the assumption that the vaccine in the coating solution and the soluble OVA label are homogenously distributed through the coating solution. Nanopatches were coated and applied to the buccal mucosa of anaesthetised mice as described above. After 2 min of application, the mouse was euthanized and the buccal mucosa was excised with the Nanopatch still held in place by the clip applicator to avoid accidental loss of antigen during excision. The Nanopatch was then removed from the mucosa and the buccal surface swabbed 5 times with a moist cotton bud to remove and quantify antigen on the surface of the mucosa. The used Nanopatch, swab and excised cheek were placed into individual scintillation vials. Cheek tissue was treated with 1 ml of tissue solubiliser solution (Solvable, Perkin-Elmer, Massachusetts, USA) at 60 °C overnight. Nanopatches and swabs remained at room temperature overnight in 1 ml PBS. Ten millilitres of scintillation liquid (Ultima Gold, PerkinElmer, Massachusetts, USA) was added to each vial and the samples were vortexed vigorously prior to counting in a scintillation counter for 4 min per sample. Skin delivery of Fluvax was quantified in a similar way after application of Nanopatches to the ear, as described previously [13]. Eight replicates of both skin and mucosa samples were quantified and compared with coated Nanopatches (n = 4). 2.6. Penetration depth evaluation To measure the location to which the Nanopatch would deliver vaccine, Nanopatches were coated using 0.2 μm FluoSpheres® (Invitrogen Australia Pty Ltd, Mulgrave, VIC, Australia) prior to application to the mucosa. These were applied using the methods above, on live, anaesthetised mice. Following application the mice were sacrificed using cervical dislocation and their full cheek was excised. This was then fixed using 4% paraformaldehyde solution in phosphate buffered saline (PBS) for 1 h. The cheek tissue was then embedded in TissueTek® OCT compound (ProSciTech, Thuringowa, QLD, Australia) within a cryo mould and frozen using liquid nitrogen. These blocks were then sectioned using a cryostat (Leica Microsystems, North Ryde, NSW, Australia) at 10 μm thickness, with areas selected randomly across the patched area. From each buccal mucosum 30–40 sections were cut and mounted onto glass slides by the HistoTechnology facility at the QIMR Berghofer Research Institute. These were imaged at 20× with a 488 nm laser with an absorbance of 500–550 nm using a Zeiss LSM510 Meta multi-photon microscope (Carl Zeiss Inc, Germany).

From the samples a total of over 100 projection depth measurements were made, which is sufficient to give confidence in the overall population results [31]. 2.7. Cryo-SEM To observe the overall penetration of the surface of the mucosa it was desirable to observe the buccal mucosa tissue in-situ. To do this, Nanopatches were applied to the buccal mucosa as described above with the tissue being removed with the patches maintained in place. Following this, the whole Nanopatch and tissue assembly was frozen in slush liquid nitrogen and then removed under vacuum. Once in a cryo-preparation chamber and under vacuum, the Nanopatch was removed, whilst maintaining the tissue at − 180 °C. Finally, this tissue was sputter coated with a few nanometres layer of gold and then imaged using a Philips XL30 scanning electron microscope (Philips, Netherlands). 2.8. Evaluation of APC targeting Nanopatches coated as described above containing the active agent DiD (Invitrogen Australia Pty Ltd, Mulgrave, VIC, Australia) were used for the evaluation of APC targeting. The Nanopatches (n = 4) were applied to the buccal mucosa of anaesthetised MacGreen mice for 2 min before the animal was euthanized and the cheek was excised. Tissue was fixed in 2% paraformaldehyde for 60 min and stored in PBS before imaging by confocal microscopy on a Zeiss LSM510 Meta (Carl Zeiss Inc, Germany). A total of 10 areas of mucosa, 0.1 mm2 in size were scanned to a depth of 100–150 μm and analysed for both sites of antigen delivery and enumeration of dendritic cells. 2.9. Immunisation and sampling For each group of the immunisation study, n = 5 animals were used. Each animal received a dose of 37 ng of Fluvax2010® at Day 0 and Day 28. A single Nanopatch was applied as described to the ear or inside cheek of mice at each immunisation. Oral delivery of vaccine was used to control for any vaccine washed off the Nanopatches and into the GI tract. To do this mice were lightly anaesthetised with methoxyflurane (Ceva Delvet Pty Ltd, Seven Hills, NSW, Australia) and sterile, 20 gauge plastic feeding tubes (Instech Laboratories, Inc., Pennsylvania, USA) were used to orally gavage the mice with 100 μl Fluvax2010® vaccine solution. For intra-muscular administration of the vaccine, mice were also lightly anaesthetised with methoxyflurane and 30 μl of vaccine solution was delivered to each quadriceps muscle of the hind leg of the mouse using a 29 gauge insulin syringe (BD Biosciences, North Ryde, NSW, Australia). Blood was collected from the retro-orbital venous sinus under methoxyflurane anaesthesia and the serum was extracted by centrifugation. Reproductive tract samples were collected by gently pipetting 100 μl of sterile PBS containing protease inhibitors (SigmaAldrich Pty. Ltd., Sydney, NSW, Australia) in and out of the vaginal cavity of mice five times. Fresh faecal pellets were collected, weighed and added to PBS containing protease inhibitors to a concentration of 100 mg faeces/ml. Faecal samples were vigorously vortexed to disrupt the solid matter, incubated at 4 °C for an hour, re-vortexed then centrifuged and the supernatant transferred to a fresh eppendorf tube for analysis. Upper respiratory tract samples were collected after euthanasia of the mice by cervical dislocation. After euthanasia an incision was made at the neck to expose the trachea and the trachea clamped. The mouse was positioned with the head tilting downwards and a 25 gauge needle attached to a 1 ml syringe containing 0.5 ml PBS/protease inhibitors inserted into the trachea above the clamp. The PBS/protease inhibitor solution was slowly injected into the trachea and the fluid draining from the nose was collected into an eppendorf tube positioned over the nose of the mouse. All samples were stored at − 20 °C until required.

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2.10. Serological analysis ELISAs were performed as previously described [4]. Briefly, the trivalent influenza vaccine Fluvax 2010® was diluted to a concentration of 3 μg HA protein/ml in 0.1 M sodium bicarbonate buffer for coating. Each well of 96 well Nunc Maxisorb plates (Thermo Fischer Scientific Australia Pty Ltd, Scoresby, VIC, Australia), was coated with 50 μl diluted vaccine overnight at 4 °C. The plates were blocked with 4 mg/ml BSA (Sigma-Aldrich Pty. Ltd., Sydney, NSW, Australia) before addition of biological samples. In this study, serum was used at a starting dilution of 1/100 and serial doubling dilutions were made in BSA/PBS. For mucosal samples, intestinal and nasal lavage samples were used at a starting dilution of 1/2 whilst vaginal lavage samples were used at a starting dilution of 1/5. Again, doubling dilutions of mucosal samples were made in BSA/PBS. After washing, anti-mouse IgG HRP was used at 1:600 to detect IgG responses and anti-mouse IgA HRP was used at 1:1000 for the detection of IgA responses (both Invitrogen Australia Pty Ltd, Mulgrave, VIC, Australia). The substrate ABTS (Sigma-Aldrich Pty. Ltd., Sydney, NSW, Australia) was used for colour development and the reaction was stopped with the addition of 50 μl 1% SDS. The absorbance was read at 405 nm on a Fluostar Omega BMG Labtech reader with Omega software version 1.30. Endpoint titres were defined as the reciprocal of the highest dilution of samples that yielded an optical density at 405 nm of more than 3 standard deviations above the mean optical density of control samples obtained from naive mice. 2.11. HI assay Sera was treated for nonspecific hemagglutination inhibitors with receptor destroying enzyme (RDE (II) Denka Seiken Co., Ltd, Tokyo, Japan) using a 1:3 ratio and heated for 18 h at 37 °C, followed by RDE neutralisation at 56 °C for 1 h. Sodium citrate (1.6%) was then added to the sera making a final dilution of 1:10. Hemagglutination Assay (HA) was carried out to determine the endpoint titration of virus stock (inactivated A/Perth/16/2009 HA, antigen supplied by the Victorian Infectious Diseases Reference Laboratory, North Melbourne, Australia). This was done by performing two-fold serial dilutions of the reference virus and adding an equal volume each of PBS and 1% chicken red blood cells (CRBCs; Australian SPF Services Pty. Ltd., Woodend, VIC, Australia). After 30 minute incubation at room temperature one Hemagglutination Unit (1 HAU) was determined as the highest viral dilution to completely agglutinate the CRBCs. The Hemagglutination Inhibition (HI) assay was performed by making two-fold serial dilutions of serum (25 μl final volume), then adding 25 μl of 4 HAU virus stock to all sera wells or 25 μl of PBS to control wells. The plate was tapped gently to mix solutions, and then incubated at RT for 30 min. Following incubation at room temperature for 30 min, 25 μl of 1% CRBC was added to all wells and incubated for a further 30 min at room temperature. The HI titre was then established by the final sera dilution to completely inhibit the agglutination of CRBCs. 2.12. Statistics Statistical analyses were done in GraphPad Prism v6.02 software. 3. Results 3.1. Development of a clip applicator for application of Nanopatches to the buccal mucosa Our clip applicator designed specifically for these experiments fitted well within the mouth of a mouse (when anaesthetised) with the Nanopatch. Our application method involved placing the device in the mouth of the mouse and then releasing the clip which provided a slow dynamic application—in the order tens of centimetres per second.

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The resulting static force that the clip maintained on the skin was measured using a load cell to be 1.9 Newtons (+/−8% SD). We then evaluated the payload delivery into the mucosa, with the goal of achieving consistent, reproducible penetration and accurate payload delivery. To achieve this we applied Nanopatches coated with fluorescent FluoSpheres® to the buccal mucosa for analysis of penetration depth (Fig. 2A–D). Specific distinctions in the layered structure of the buccal mucosa were defined by the presence of collagen fibres in the lamina propria, which emit a second harmonic signal visualised in unstained tissue sections by multi-photon microscopy. Our projections were found to reach a depth of 47.8 ± 14.8 μm (mean ± SD, 4 mouse buccal mucosae) (Fig. 2E) from the mucosal surface to the distal tip of the Nanopatch projections. We observed that the keratinized layer of the mouse buccal mucosa (reported to be 10–15 μm) was breached resulting in delivery of the payload to the epithelium (down to around 50 μm depth), lamina propria and into the upper sub-mucosa of the mouse mucosa. To image the effect of Nanopatch penetration on the mucosal tissue surface CryoSEM was employed. We then observed the resulting matrix of residual Nanopatch projection holes in the mucosa and mucosal texture (Fig. 2F). As the experimental method does not remove the Nanopatch until it is frozen with the tissue, the results are effectively those seen in-situ. Restrictions of space within the mouse mouth create difficulties in precisely placing Nanopatches fully flat on the buccal mucosa. We estimate that at least two thirds of projections were within the mucosa during an application. To quantify the amount of vaccine that was delivered, Nanopatches coated with the seasonal influenza vaccine Fluvax2010® and a C14 labelled ovalbumin tracer were applied to the buccal mucosa of mice. The mucosa was then swabbed to remove any payload that remained on the surface to differentiate between payload that was likely delivered into the oral cavity and thereby ingested and that which was delivered into the mucosa. The measured delivery efficiency of vaccine was 31.7 ± 3.7% into the mucosa with 9.0 ± 3.4% remaining on the tissue surface (mean ± SD, n = 8) (Fig. 2G). Importantly, there were no macroscopically visible effects such as bleeding or edema on the mouse oral cavity immediately after Nanopatch application or for the duration of the experiments (10 weeks). Mice immunised at the buccal mucosa were seen to consume food and water as normal, no weight loss was observed and no changes in behaviour were noticed. 3.2. Targeting of APCs in the oral mucosa Having established application conditions and verified penetration depth and payload delivery into the mouse buccal mucosa, payload delivery to the APCs was then evaluated. We confirmed the targeting of mucosal APCs by buccal Nanopatch application using a lipophilic dye (DiD) as a marker for vaccine delivery location and applied to the cheeks of transgenic MacGreen mice (n = 4) in which all macrophage derived cells, including Langerhans cells and other dendritic cell subsets, express eGFP (Fig. 3). The average number of projection sites observed to penetrate in each 0.1 mm2 area of buccal mucosa was 21.8 ± 9.5 (n = 10). In the same area, an average of 49.7 ± 22.5 dendritic cells were visualised, based on both the expression of eGFP and dendritic cell morphology, of which 6.7 ± 4.1 (13%) cells colocalised with a site of dye deposition. This represents approximately 6.5% of the total area of mucosa targeted by the Nanopatch. This method has previously been used to identify the colocalisation of vaccine in skin vaccination using microprojections [4,24]. If these figures are extrapolated to the area targeted by a single Nanopatch applied to the mucosa (approximately two thirds of projections penetrating), the number of dendritic cells targeted by Nanopatch application would be approximately 5110 with 690 dendritic cells co-localising with sites of dye (or vaccine) deposition; per patch applied to the mucosa. These data confirm the capability of the Nanopatch to place antigen specifically in

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Fig. 2. Penetration into mucosa and release of active agent. A representative section of buccal mucosa after application with a Nanopatch coated with Fluospheres® showing (A) transmitted light image (B) confocal image of fluorescent Fluospheres® (C) collagen in the lamina propria detected by second harmonic signals using multi-photon microscopy (D) merged image of (A), (B) and (C) showing depth penetration of Nanopatch in the buccal mucosa. Scale bar on A–D is 50 μm. (E) Penetration depth measurements in the buccal mucosa (n = 4 mice with total 136 penetration holes measured). (F) Cryo-SEM image of the buccal mucosa after Nanopatch application showing clear penetration through the keratinised layer of the mucosa and penetration into the epithelium and lamina propria. (G) Percentage of vaccine (Fluvax® 2010) detected within the buccal mucosa and on the surface of the mucosa using radiometric methods (n = 4 mice were used to assess delivery).

interstitial space that is rich in APCs and likely to induce rapid antigenic uptake. 3.3. Evaluation of immune responses following immunisation with a conventional influenza vaccine (split virus) Mice (n = 5) were immunised with 37 ng of the split virus seasonal influenza vaccine (Fluvax®2010) at day 0 and a booster immunisation delivered at day 28. Four delivery methods of immunisation were compared: intra-muscular immunisation with needle and syringe as a

Fig. 3. Representative (A) low (×10) and (B) high (×40) magnification compressed Zstack image of MacGreen mouse buccal mucosa after application of a Nanopatch coated with a formulation containing DiD. Dendritic cells (green) are characterised by the expression of eGFP and the typical dendritic cell morphology. Arrows in B indicate the colocalisation of two DiD (red) deposition sites with the dendrites or cell body of dendritic cells. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

conventional immunisation delivery route; application of a single vaccine coated Nanopatch to the buccal mucosa; application of a single Nanopatch to the ear skin as this has previously been shown to be capable of inducing systemic immune responses with low amounts of vaccine [4]; and oral delivery of the vaccine by gavage tube to the stomach. The oral delivery group is a conventional mucosal immunisation route, but also acts as a control group for any vaccine that may be ingested following application of the Nanopatch to the buccal mucosa. Total serum IgG responses were evaluated 21 days after the initial immunisation (Fig. 4A). The mice that received the immunisation by Nanopatch application to the buccal mucosa showed a significant

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comparative mucosally immunised mice that received the vaccine orally at both 2 weeks (p = 0.0666) and 6 weeks (p = 0.0016) after immunisation. This confirms that the serum IgG response seen in bucally immunised mice is due to uptake of vaccine in the oral mucosa and not as a result of ingestion of the vaccine. At both 2 and 6 weeks after the booster immunisation, mice that received the immunisation at the buccal mucosa show no significant difference in serum IgG titre when compared to mice that received the immunisation to the skin (p = 0.8447 and p = 0.1008 respectively) or intramuscularly (p = 0.1505 and p = 0.1101 respectively). As mucosal routes of immunisation have been reported to induce IgA production, lavage fluid from the reproductive tract (female mice were used in this study), supernatant from faecal pellet preparations and upper respiratory tract lavages were evaluated for IgA 6 weeks after the booster immunisation. In all groups the level of IgA was low and variable, and no significant differences in the level of IgA in bucally immunised mice with other routes of immunisation were detected. The only group to show significantly increased levels of IgA were the mice that received the immunisation by Nanopatch to the skin. This group of mice had significantly higher levels of intestinal IgA when compared with all other groups (p b 0.01 in all cases). The haemagglutination inhibition (HI) assay was used to analyse the functional relevance of the serum IgG response at Day 21 against the H3N2 A/Wisconsin/15/2009 component of the vaccine. A HI titre of N40 is considered the minimum that correlates with protection in humans (indicated on the graph by a black line). Eighty percent (4/5) of the mice that received the immunisation with the Nanopatch at the buccal mucosa exceeded this minimum HI level. As expected, given the lack of immune response detected in the ELISA assays from orally immunised or naïve mice, no HI activity was detected in the sera from these mice. All of the mice that were administered the vaccine by Nanopatch to the skin exceeded the HI threshold, whilst only a single animal that received the immunisation intra-muscularly reached this minimum value. The HI responses detected in the Nanopatch immunised groups after a single immunisation indicate that for functional systemic immune responses a booster immunisation is not required when the vaccine is administered by these routes. 4. Discussion

Fig. 4. Anti-influenza immunogenicity of Fluvax® 2010 coated Nanopatches applied to the buccal mucosa. (A) Total serum IgG responses at various times after immunisation and boost. (B) IgA responses in samples extracted from faeces, female reproductive tract and nasal passages after the final immunisation (Day 70). (C) Haemagglutination inhibition titres against the A/Perth/16/2009 like influenza strain in serum collected 21 days after the initial immunisation. The black line at a HI titre of 40 represents the accepted minimum that correlates with protection in humans. In all panels bars represent the mean and standard deviation from the mean are shown. Significance of p ≤ 0.01 (***), p = 0.01–0.05 (**) or p = 0.05–0.1 (*) is indicated. In all immune studies n = 5 animals were used per group.

increase in the mean total serum IgG titre when compared with the mice that received the vaccine by the oral route (p = 0.0007). Mice that received the vaccine by Nanopatch application to the ear skin showed the strongest serum IgG response, significantly higher than mice that received the immunisation by any other route (all p b 0.0001). A booster immunisation of 37 ng Fluvax®2010 was delivered by the same route at Day 28 and total serum IgG measured at 2 and 6 weeks after this immunisation (Fig. 4A). The serum IgG response in mice immunised at the buccal mucosa was significantly higher than the

Direct delivery of vaccine to mucosal surfaces has the potential for eliciting protective immune responses at their surface, for an efficient first line of defence against infiltrating antigens. For the first time, in this study we present a simple, easily applied mechanical device to generate a systemic immune response to an antigen with buccal mucosa immunisation—aimed at directly reaching large populations of immune cells. Specifically, this paper aimed to introduce a method that would be acceptable in clinical practice without significant discomfort or pain. The clip application of a Nanopatch serves to do this, with successful systemic immune response generation. The following sections discuss the mechanical challenges that we have overcome by this technology, and the subsequent immunological potential. 4.1. Nanopatch application to buccal mucosa using a clip applicator Our application of Nanopatches to the buccal mucosa in mice successfully introduces a vaccine (or potentially another bio-therapeutic) to a precise anatomical location. The surface of the mucosa, shown in Fig. 2F, clearly indicates that the projections from our device have entered into the mucosa. Whilst the saliva layer on the surface of the tissue was potentially an additional barrier to effective delivery into the mucosa tissue, the low mass fraction of vaccine remaining on the mucosal surface—compared to that delivered into the mucosa—shows there appears to be no detrimental effect due to the its presence. Indeed, this observation is further supported by comparison with Nanopatches applied to relatively dry skin [13]—which had a similar mass fraction

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deposited on the surface. It is possible that the potential effect of the buccal saliva on delivery is mitigated by the rapid application of the Nanopatch to the surface—with only milliseconds of time for potential diffusion (application time is investigated by [32] and diffusion rates in skin is studied in [33]). The varied surface texture shown in Fig. 2F indicates that there are different levels of mucous which may however change the ability of projections to penetrate. One consideration of this is that in human clinical studies the projections may have to be significantly longer to overcome the thicker human mucous layers. The observed consistent and repeatable skin surface penetration of projections (Fig. 2F) was supported by the delivery of FluoSpheres® into the tissue (Fig. 2A–D). The location of antigen deposition in this study has shown that projections that penetrate the murine buccal mucosa will be reaching the antigen presenting cells that are located within the mucosal epithelial layer and lamina propria. Whilst this will be useful if the immune response is mediated by Langerhans cells (the only APCs residing in the mucosal epithelium), if dendritic cells deeper in the mucosa (i.e. the lamina propria) are desired targets for particular immune responses, then deeper penetration or a highly diffusive formulation may be preferred. Due to the large size of the patch (4 x 4 mm)—compared with the size of a mouse mouth—there was difficulty ensuring that all projections penetrated the tissue. Instead, we expect that around two-thirds of projections entered the tissue, with the last third being mis-aligned and reducing their performance. This result was significantly less than the ~ 90% of Nanopatch projections penetrating mice ear skin [13] and flank [34] in which there was more scope for alignment of the patch with the tissue surface. Here, we sought to mitigate the differences between the number of projections penetrating the mucosal tissue and mouse ear by matching the delivered doses (between the two sites). Nevertheless, there are clear differences in the number and type of APCs targeted between the skin and mucosal targeting of the Nanopatch (discussed below, in 4.2). Whilst the mechanical penetration of the tissue in this work has achieved a good proof of concept, human buccal mucosa tissue has previously presented a much greater challenge. Specifically, it has a layer of salivary film in the order of 700–100 μm [35], mucosal epithelial of around 500 μm thick [36] and a sub-mucosa below this containing collagen and blood vessels. These thicknesses are, however, only approximate as the regions where these layers join are much more variable than in mouse tissue. Despite this, there are some potential benefits to application of Nanopatches to human tissue: 1. Human tissue does not have a keratinised layer that will have restricted penetration in the mouse; 2. Humans have a far larger area in which to fit Nanopatches and associated application devices; and 3. Humans will be more agreeable to this method of application; Furthermore, if we fabricate longer projections (in the region of hundreds of micrometres long) then these should puncture to the appropriate regions in human tissue. Rapid release of the vaccine into the buccal mucosa (if replicating the 2 min achieved in this study (Fig. 2))—also means that, in a clinical setting, there would be minimal risk of choking, dislodgement or swallowing of the device as the Nanopatch is maintained in place for the duration of the vaccination by the applicator. Indeed the device is small enough that it is unlikely to obstruct a human airway even if it were to be swallowed. Our previously published work on skin has shown success in puncturing a hard surface, so the softer tissue of the buccal mucosa should be easily overcome for clinical translation. 4.2. Immunological targeting of vaccine to antigen presenting cells within the buccal mucosa for systemic immune responses Buccal dendritic cells have been shown to have antigen presenting cell (APC) functions [37] and induce specific immune responses including the generation of antibody responses [38] and CD8+ CTL immunity

[39]. Recently, unique populations of dendritic cell subsets were identified within the buccal mucosa, analogous to skin dendritic cell subsets, although these were found to have T-cell priming activities different to that seen in skin [40]. Whilst in the field of microneedle immunisation where antigen is targeted to dendritic cell populations the relative roles of these cell subsets remains to be fully elucidated, it is tempting to speculate that the difference in immune response seen in our study between skin and mucosally immunised groups is due to the number, and perhaps subsets of dendritic cells reached. Fernando et al. [4] showed that a single Nanopatch applied to the skin of a mouse deposited antigen to approximately 4500 LCs and 2500 dDCs in the skin (approx 7000 DCs in total), whilst here, our buccal application of the same Nanopatch configuration delivered antigen in the vicinity of only ~ 700 LCs and DCs of the buccal mucosa. Indeed, to extend this conjecture, the levels of total IgG detected in serum after a single immunisation where skin N mucosal N muscular also reflect the number of DCs targeted by these immunisation routes (7000 DCs N 700 DCs N relative paucity). This observation may indicate that the initial availability of APCs at the site of immunisation to uptake and shuttle antigen to local lymphoid tissue may be a critical factor in the initiation of subsequent immune responses, particularly for very low doses of antigen such as those in this study. In comparison to our physical mucosal delivery method, other published immunological studies delivering antigen to the buccal mucosa have relied on passive uptake of antigen by APCs after topical application or delivery into the buccal mucosa with needle and syringe [38,39]. Neither of these methods specifically target antigen to the mucosal dendritic cells. Topical application of antigen is to some extent, indiscriminate as it does not directly deliver antigen to APCs, but relies on vaccine antigen being captured within mucous and subsequent uptake by immune cells. Needle and syringe delivery is an imprecise methodology that likely delivers vaccine deeper than the lamina propria and into the underlying musculature (given that the bevel length of most needles exceeds 1 mm), missing many of the buccal DCs. We are currently developing alternative Nanopatch configurations that have the potential to increase the number of DCs targeted within the buccal mucosa with the goal of enhancing specific immune responses. We believe that this may facilitate the induction of IgA at mucosal surfaces that was not detected following buccal immunisation here, but is the predominant antibody at mucosal surfaces that provides a first line of defence against invading pathogens. Although the vast majority of vaccines are delivered intramuscularly by needle and syringe, two routes of mucosal immunisation are also licenced for specific vaccines. The type of antigen appears to be important in the mucosal delivery of vaccines, with particulate antigens the most effective in generating immune responses after mucosal delivery [41,42]. Flumist® is an intra-nasally delivered anti-influenza vaccine (currently only licenced for a specific age population); and, the intranasal vaccine delivery route has also raised safety concerns [43]. Several vaccines are currently licenced for oral delivery including the typhoid, cholera, poliomyelitis and rotavirus vaccines—all attenuated vaccines. However, with these exceptions, generally vaccines cannot be delivered via the oral route as the vaccines (proteins, peptides, subunit vaccines) are degraded in the gastrointestinal system [44]. Here, our device delivers a consistent, reproducible vaccine dose to mucosal surfaces capable of inducing specific immune responses and with potential to deliver a range of vaccine types. 5. Conclusions In this paper we report on the development of a novel vaccine delivery approach using a micro-device for precise targeting to a highly immunologically relevant tissue—the buccal mucosa. By utilising this novel vaccine delivery route, antigen placed in the vicinity of immune cells can elicit a systemic immune response and with further refinement may have the potential to generate a first line of defense in mucosal IgA

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for enhanced protection. Overcoming the challenges of delivering vaccine into this tissue, we showed that a simple clip applicator could enable a vaccine coated Nanopatch to elicit a strong systemic antibody response with a commercial influenza vaccine. This response was greater than that of a comparable intramuscular delivery—indicating the future potential of this delivery route and approach. Further, we measured the location that the projections reached to—and released vaccine—in the buccal mucosa and found that they deliver to a position where a large number of Langerhans and other dendritic cells reside. Indeed, overall a surface examination of the buccal mucosa showed high levels of surface penetration, despite the presence of a surface mucous layer. At this point, this mucosal targeting approach has not generated a strong mucosal IgA immune response. This may be due to: the vaccine structure (split instead of full particle [shown to generate mucosal IgA responses [42]]); the low dose (37 ng) delivered; or the number/type of dendritic cells targeted. This will be examined in future studies, however, the recent publication of microneedle delivery to the lip or tongue of rabbits that resulted in a mucosal IgA response further supports this theory [9]. The study differed from the research presented here in both antigen (particulate/DNA/protein compared with split virion) and the dose delivered with the Ma et al. study delivering approximately 1400–3300 times more antigen. As a proof of concept we have shown the great potential the Nanopatch has as a delivery device for immunotherapeutics to the mucosal site—this is the first time such a simple, pain free device has been used in this area. By accounting for key mechanical and immunological differences in the tissue, conceptually the mucosal Nanopatch could be extended to practical immunotherapies in people. Funding statement This research was funded by the Australian Research Council Discovery Scheme (MAFK and IHF, Grants ID# DP130100996) and the National Health and Medical Research Council of Australia (MAFK and MSR, Grants ID# APP11049906). IHF was funded in part by NIH grant 5U01CA141583. Acknowledgements The authors would like to thank all members of the Delivery of Drugs and Genes Group for this work, in particular Sally Yukiko and Ben Baker for their assistance in coating, Jacob Coffey for his assistance with microscopy and Chelsea Stewart for assistance with animal work. Nanopatches were fabricated at the Rutherford Appleton Laboratories, UK, by Derek Jenkins. This work was performed in part at the Queensland node of the Australian National Fabrication Facility, a company established under the National Collaborative Research Infrastructure Strategy to provide nano and micro-fabrication facilities for Australia's researchers. References [1] Y. Nir, A. Paz, E. Sabo, I. Potasman, Fear of injections in young adults: prevalence and associations, Am. J. Trop. Med. Hyg. 68 (3) (2003) 341–344. [2] Minimising the risk of syringe reuse Unicef, http://www.unicef.org/supply/index_ 55205.html2012 (updated 25 May 2012; cited 2012, Available from). [3] X. Chen, G.J. Fernando, M.L. Crichton, C. Flaim, S.R. Yukiko, E.J. Fairmaid, et al., Improving the reach of vaccines to low-resource regions, with a needle-free vaccine delivery device and long-term thermostabilization, J. Control. Release 152 (3) (2011) 349–355. [4] G.J. Fernando, X. Chen, T.W. Prow, M.L. Crichton, E.J. Fairmaid, M.S. Roberts, et al., Potent immunity to low doses of influenza vaccine by probabilistic guided microtargeted skin delivery in a mouse model, PLoS One 5 (4) (2010) e10266. [5] M. Kendall, Engineering of needle-free physical methods to target epidermal cells for DNA vaccination, Vaccine 24 (21) (2006) 4651–4656. [6] C. Czerkinsky, J. Holmgren, Topical immunization strategies, Mucosal Immunol. 3 (6) (2010) 545–555. [7] J. Holmgren, C. Czerkinsky, Mucosal immunity and vaccines, Nat. Med. 11 (4 Suppl.) (2005) S45–S53.

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Microprojection arrays to immunise at mucosal surfaces.

The buccal mucosa (inner cheek) is an attractive site for delivery of immunotherapeutics, due to its ease of access and rich antigen presenting cell (...
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