Transcending epithelial and intracellular biological barriers; a prototype DNA delivery device

Journal of Controlled Release 226 (2016) 238–247

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Transcending epithelial and intracellular biological barriers; a prototype DNA delivery device Joanne McCaffrey, Cian M. McCrudden, Ahlam A. Ali, Ashley S. Massey, John W. McBride, Maelíosa T.C. McCrudden, Eva M. Vicente-Perez, Jonathan A. Coulter, Tracy Robson, Ryan F. Donnelly, Helen O. McCarthy ⁎ School of Pharmacy, Queen's University Belfast, 97 Lisburn Road, Belfast BT9 7BL, Northern Ireland, UK

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Article history: Received 10 November 2015 Received in revised form 10 February 2016 Accepted 11 February 2016 Available online 13 February 2016 Keywords: Microneedle Gene delivery Gene therapy Cell penetrating peptide DNA nanoparticle

a b s t r a c t Microneedle technology provides the opportunity for the delivery of DNA therapeutics by a non-invasive, patient acceptable route. To deliver DNA successfully requires consideration of both extra and intracellular biological barriers. In this study we present a novel two tier platform; i) a peptide delivery system, termed RALA, that is able to wrap the DNA into nanoparticles, protect the DNA from degradation, enter cells, disrupt endosomes and deliver the DNA to the nucleus of cells ii) a microneedle (MN) patch that will house the nanoparticles within the polymer matrix, breach the skin's stratum corneum barrier and dissolve upon contact with skin interstitial fluid thus releasing the nanoparticles into the skin. Our data demonstrates that the RALA is essential for preventing DNA degradation within the poly(vinylpyrrolidone) (PVP) polymer matrix. In fact the RALA/DNA nanoparticles (NPs) retained functionality when in the MN arrays after 28 days and over a range of temperatures. Furthermore the physical strength and structure of the MNs was not compromised when loaded with the NPs. Finally we demonstrated the effectiveness of our MN-NP platform in vitro and in vivo, with systemic gene expression in highly vascularised regions. Taken together this ‘smart-system’ technology could be applied to a wide range of genetic therapies. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Advancements in molecular biology and biotechnology have resulted in the progression of pharmaceutical agents from traditional low molecular weight drugs to biomacromolecules such as DNA, RNA, peptides and proteins [1]. In particular, the administration of DNA as a pharmaceutical agent for the prevention or treatment of disease has garnered intense research over the past 20 years [2]. In theory, DNA delivery has the potential to revolutionise the treatment and management of a range of pathological conditions. As such, there has been a concerted effort to develop effective and clinically applicable gene delivery systems. However, despite early encouraging results there has been a failure to realise the full potential of gene therapy in vivo, particularly in clinical trials. The void in patient translation can be attributed to the numerous physiological barriers that must be overcome by the DNA even before optimal therapeutic translation occurs [3]. Such barriers include first pass metabolism, degradation by serum endonucleases, clearance by immune cells, traversing cellular membranes and degradation in the cellular cytosol [4]. Therefore for significant advancements in the field of gene therapy, improvements in delivery technologies and pharmaceutical formulation have become an absolute necessity [5]. ⁎ Corresponding author. E-mail address: [email protected] (H.O. McCarthy).

http://dx.doi.org/10.1016/j.jconrel.2016.02.023 0168-3659/© 2016 Elsevier B.V. All rights reserved.

The skin represents an ideal target for the delivery of DNA, with highly developed vascular and lymphatic networks and rich populations of antigen presenting cells [6]. Delivery of DNA into the skin layers can be employed for the treatment of dermal diseases, cutaneous cancers, wound healing and vaccination with the added advantage of circumventing many barriers associated with parenteral delivery [7]. Transdermal delivery enables localised and/or systemic dissemination of the DNA, avoids first pass metabolism clearance and confers a large surface area target [8]. However, the rate-limiting factor for transdermal delivery lies in the biological barrier of the outermost layer of the skin, the stratum corneum (SC). A number of physical delivery strategies have been employed for the transport of DNA across the SC, such as, electroporation [9], gene gun [10], liquid jet injection [11], and tattooing [12]. Frequently, these methods involve the use of expensive equipment, specialist training and trained health care professionals. A more recent innovative strategy to overcome the SC barrier is the use of microneedle (MN) technology [13]. MN arrays comprise a backingplate with numerous microprojections designed to create transient pores through the SC. The transient pores provide a window for the delivery of the cargo directly by-passing the SC [14]. Strategies for DNA delivery by MN include SC disruption followed by topical application of a DNA solution onto the compromised skin [15], using DNA-coated stainless-steel solid MNs to deposit DNA in the skin [16], and DNAincorporating dissolving polymeric MNs that discharge their cargo as

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they dissolve [17]. The use of dissolving MN arrays encapsulating the cargo within the matrix can overcome some of the limitations associated with the use of solid MN arrays with respect to limited loading capability, scalability, manufacturing costs and the need for sharps disposal following use. However to date, the majority of MN studies have focused on the delivery of ‘naked’ DNA without consideration of the complex intracellular barriers associated with gene delivery. Thus, the addition of a delivery system, to overcome the intracellular barriers to gene delivery could significantly enhance the payload to the skin. We have previously described the use of an amphipathic peptide, RALA, for the nanoencapsulation of plasmid DNA; the nanoparticles are self-assembling, and are capable of eliciting gene expression following parenteral delivery in vivo [18,19]. This peptide facilitates the intracellular delivery of DNA across the cell membrane, aids endosomal escape of the DNA cargo and promotes nuclear localisation of the DNA for transcription. Herein we describe a novel MN platform that incorporates RALA/plasmid DNA nanoparticles in a dissolving polymeric formulation. The arrays were fabricated using a mechanically robust polymer, poly(vinylpyrrolidone) (PVP) suitable for low-cost manufacture of MN patches that do not compromise the transfection efficacy of the bioactive RALA/pDNA nanoparticles. The combination of these two delivery platforms resulted in the production of a state of the art technology platform for the delivery of nucleic acids. 2. Materials and methods 2.1. Materials 2.1.1. RALA peptide The RALA peptide was produced by solid-state synthesis and supplied in acetate salt as a desalted lyophilized powder (Biomatik, USA) as described in McCarthy et al. (2014). 2.1.2. Plasmid(s) pCMV-Red Firefly Luc (pCMV-Luc) was purchased from ThermoScientific (USA) and pEGFP-N1 was purchased from Clontech (USA). Plasmids were propagated in MAX Efficiency® DH5α™ Competent Cells (Life Technologies, UK), purified using PureLink® HiPure Plasmid Filter Maxiprep Kit (Life Technologies, UK) and concentration quantified by UV absorption at 260 nm using a Nanodrop (Thermo Scientific, UK). 2.1.3. Cell line(s) The NCTC-929 fibroblast cell line was obtained from the American Type Culture Collection. The cell line was authenticated by short tandem repeat (STR) profiling carried out by the suppliers and routine testing confirmed the cells were Mycoplasma-free (Plasmotest, Invivogen, France). 2.1.4. Animals Female six-week-old C57BL/6 mice were purchased from Charles River Laboratories. The animals were housed in an open facility at 21 °C and 50% humidity with food and water ad libitum. The experimental protocols were compliant to the UK Scientific Act of 1986 and project license 2678. 2.2. Methods 2.2.1. Synthesis of RALA/pCMV-Luc nanoparticles RALA/pDNA nanoparticles were prepared at N:P ratios (the molar ratio of positively charged nitrogen atoms in the peptide to negatively charged phosphates in the pDNA backbone) 4, 6, 8, 10 and 12 by adding appropriate volumes of RALA peptide solution to pCMV-Luc. The complexes were incubated at room temperature for 30 min to allow nanoparticle formation. Nanoparticles were subsequently analysed and used immediately following incubation.

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2.2.2. Particle size and zeta potential analysis RALA/pCMV-Luc nanoparticles were prepared at N:P ratios 4, 6, 8, 10 and 12, containing 1 μg pDNA. These samples were analysed for particle size in a disposable microcuvette and for zeta-potential in a disposable folded capillary cell (Malvern Instruments, UK). Samples were diluted to 1 ml with distilled water before the measurement of zeta potential analysis. Particle size and zeta potential were determined using a Nano ZS Zetasizer and DTS software (Malvern Instruments, UK). 2.2.3. Serum stability RALA/pCMV-Luc nanoparticles N:P ratio 10, encapsulating 1 μg pDNA, were prepared and incubated at 37 °C in the presence of 10% foetal calf serum for 0, 2, 6, 12 and 24 h. Following this incubation Sodium Dodecyl Sulphate (SDS) (Sigma-Aldrich, UK) was added (10%) to decomplex the nanoparticles and samples were electrophoresed through a 1% agarose gel containing ethidium bromide (0.5 μg/ml) with Tris-acetate running buffer at 80 V for 60 min and visualised using a Multispectrum Bioimaging System (UVP, UK). This study was repeated using naked pCMV-Luc as a control for comparison purposes. 2.2.4. Incubation stability study RALA/pCMV-Luc nanoparticles were prepared at N:P ratio 10 and incubated at room temperature for 0, 7, 14, 21 and 28 days, after which the particle size and zeta potential of the nanoparticles were analysed using a Nano ZS Zetasizer and DTS software. 2.2.5. Temperature stability study RALA/pCMV-Luc nanoparticles were prepared at N:P ratio 10 and the size of the nanoparticles monitored over a temperature range of 4–40 °C in 4 °C intervals using the Nano ZS Zetasizer with DTS software. 2.2.6. Manufacture of stock PVP solutions for MN fabrication PVP (MW 360,000) was purchased from Sigma-Aldrich (UK). Stock solutions of 20% and 40% w/w were prepared by adding the required mass of PVP to deionised water, followed by heating to 70 °C for 3 h, until clear, homogeneous gels were formed. Upon cooling the aqueous polymers were readjusted to 20% and 40% concentrations by addition of an appropriate amount of deionised water to account for water lost during solubilisation. 2.2.7. Agarose gel analysis of stability of pCMV-Luc ± RALA in PVP matrix RALA/pCMV-Luc nanoparticles (N:P 10), formulated as previously described, and comprising 36 μg of pDNA, were mixed in a 1:1 ratio with the 40% PVP stock solution. Following drying and solidification of the nanoparticle-loaded PVP gels at room temperature for 48 h, the gels were then dissolved in PBS and electrophoresed through a 1% agarose gel as described previously. Nanoparticle-loaded PVP gel formulations were also stored for up to 28 days at room temperature and subjected to the same agarose gel analysis to determine the long-term stability profile of the formulation. 2.2.8. In vitro PVP biocompatibility study NCTC-929 fibroblast cells were maintained in MEM (Gibco, UK) supplemented with 10% Foetal Horse Serum (FHS) (PAA, UK). Cells were maintained as monolayers, incubated at 37 °C, under 5% CO2 and subcultured every 3–4 days to maintain exponential growth. NCTC-929s were seeded at a density of 2.5 x 104 cells/well onto 96well tissue culture plates (VWR, UK) for 24 h prior to assay. Media was then supplemented with 20% PVP to concentrations of 0, 5 and 10 mg/ ml. Cells were incubated under standard cell culture conditions for 24 h following which 10% MTT reagent (Sigma-Aldrich, UK) was added to the media and cells incubated for a further 3 h. At the end of this incubation the medium was removed and DMSO added to the cells to solubilise the formazan product. Subsequently, the plates were shaken for 1 min and absorbance was measured at 570 nm on an EL808 96-well plate reader (BioTek Instruments Inc., UK). The measured

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absorbance values are expressed as a percentage of the control where the control is defined as 100% viable.

2.2.9. Transfection efficacy of RALA/pEGFP-N1 nanoparticles released from PVP matrix pEGFP-N1 and RALA/pEGFP-N1 nanoparticles at N:P ratios 4, 6, 8 and 10 were incorporated at a 1:1 ratio into the 40% PVP stock solution. The nanoparticle-loaded PVP gels were dried at room temperature for 48 h and dissolved in Opti-MEM (Gibco, UK). NCTC-929 cells were seeded at a density of 1 x 105 cells/well onto 24-well tissue culture plates (VWR, UK) for 24 h prior to transfection. Cells were conditioned for 2 h in serum-free Opti-MEM media which was then supplemented with the dissolved polymeric solution, which was equivalent to 2 μg pEGFP-N1 per well. Following incubation for 6 h the media was removed and replaced with serum supplemented culture media. Transfection was repeated with pEGFP-N1 and RALA/pEGFP-N1 nanoparticles N:P ratio 10 incorporated at a 1:1 ratio into the 40% PVP stock solution and stored at 4, 20 and 37 °C for 28 days to determine the long-term functionality of the nanoparticles in formulation. 2.2.10. Microscopic and flow cytometric analysis of transfection efficacy of RALA/pEGFP-N1 nanoparticles following release from PVP matrix 48 h post transfection NCTC-929 cells were imaged using an EVOS FL Cell Imaging System (LifeTechnologies, UK) using a 10× magnification. Subsequently, cells were trypsinized, resuspended in 2% formaldehyde (Gibco, UK) and stored at 4 °C until analysis could be performed. The BD FACSCalibur™ system (BD Biosciences, UK) was used for the detection of cells expressing GFP. Data was analysed using BD CellQuest™ software (BD Bioscience, UK).

2.2.11. MN array fabrication Laser-engineered silicone micro-moulds were used in manufacturing MN arrays and were fabricated using a previously described method [20]. The arrays were composed of 361 (19 × 19) needles perpendicular to the base, 600 μm in height, with a base width of 300 μm and interspaced at 50 μm. MN arrays composed of PVP, with no cargo, were prepared by loading 0.5 g 20% PVP stock solution into the silicone MN moulds, before centrifugation at 3000 g for 15 min. MN arrays containing pCMV-Luc were prepared by manually incorporating 36 μg pDNA into the 40% PVP stock solution at a 1:1 ratio. Subsequently, 25 mg of the DNA-PVP mixture was weighed into the centre of each micro-mould and centrifuged for 5 min at 3000 g. Following this, the DNA-polymer mixture was redistributed into the centre of the mould and centrifuged again for 5 min. This forms the ‘needles’ of the MN array. A further 500 mg of ‘empty’ 20% PVP was used to overlay the needles in the micro-mould, and the composite was centrifuged for a further 5 min to form the ‘baseplate’ of the array. MN arrays containing RALA/pCMV-Luc nanoparticles, containing 36 μg pDNA, were prepared by manual incorporation at a 1:1 ratio into the 40% PVP matrix. Approximately 25 mg of the nanoparticle-PVP mixture was weighed into the centre of each micro-mould and the centrifugation steps performed as previously described. Following centrifugation, all MN arrays were dried in the moulds under ambient conditions for 48 h. The MN arrays were then removed from the silicone moulds and the excess ‘side-walls’ removed [20]. 2.2.12. Scanning electron microscopy (SEM) of RALA/pCMV-Luc nanoparticle-loaded MN arrays MN arrays containing RALA/pCMV-Luc nanoparticles were fabricated as described in Section 2.2.11 and mounted into metal stubs, sputter coated with gold and allowed to dry overnight. Arrays were visualised and imaged using a Jeol JSM-840 A scanning electron microscope (Jeol, UK), magnification ×20.

2.2.13. Mechanical testing of polymeric MN arrays containing DNA and nanoparticle cargo PVP MN arrays were subjected to mechanical testing to assess MN strength and determine if loading of cargo adversely affected the integrity of the MNs. ‘Empty’, pCMV-Luc and RALA/pCMV-Luc nanoparticleloaded MN arrays were fabricated as described in Section 2.2.11 and compressed at a force of 45 N per array, using a TA-XT2 Texture Analyser (Stable Microsystems, Haslemere, UK) [20]. This strength testing was also carried out on arrays which had been stored for 28 days at 4, 20 and 37 °C to assess if the strength of the needles is affected by these storage conditions. MN arrays were visualised and imaged prior to, and post compression using a Leica EZ4D Digital Microscope (Leica Micosystems, U.K). Images were used to calculate ‘Percentage height reduction’ of the MNs through analysis in Image J software. 2.2.14. Quantification of DNA encapsulated in MN array and side-walls loaded with pCMV-Luc and RALA/pCMV-Luc nanoparticles In order to determine the quantity of DNA present in the MN array, and that lost in the excess ‘side-wall’ which is removed prior to MN usage, a PicoGreen® quantification assay was employed [21]. MN arrays loaded with 36 μg pCMV-Luc and RALA/pCMV-Luc nanoparticles containing 36 μg DNA were fabricated as described in Section 2.2.11. Following removal of the side-walls, the remaining MN array and the side-walls were dissolved separately in Tris buffer (20 mM) for 2 h. Samples of these dissolved components were plated out in 50 μl triplicates in 96 well plates and incubated with proteinase K (0.1 mg/ml) (Sigma-Aldrich, UK) at 37 °C for 1 h for lysis of the RALA/pDNA nanoparticles. Quant-iT™ PicoGreen® Reagent (Life Technologies, UK) was subsequently added to the wells and the samples analysed using a Synergy 2 Multi-Mode Microplate Reader (BioTek Instruments Inc., UK). 2.2.15. Ex vivo delivery of DNA cargo from RALA/pCMV-Luc-loaded dissolving PVP MN arrays The diffusion of RALA/pCMV-Luc nanoparticles from dissolving PVP MN arrays across dermatomed neonatal porcine skin (300 μm), a model for human skin [22], was investigated using Franz diffusion cells (Crown Glass Co., USA). The nanoparticle-loaded MN array was placed on top of the porcine skin and inserted using an applicator applying 11 N force across the base of the array. The MN-containing ‘donor’ compartment of the apparatus was clamped onto the ‘receiver’ compartment to form a closed system. The receiver compartment contained Tris buffer (20 mM), pre-warmed and maintained at 37 °C prior to commencement of the study. At predetermined time points 200 μl samples were removed from the receiver compartment and 200 μl of fresh Tris buffer added to maintain constant conditions. Samples were plated out in 50 μl triplicates and analysed by Quant-iT™ PicoGreen® dsDNA assay as described in Section 2.2.14. 2.2.16. Insertion of RALA/pCMV-Luc nanoparticle-loaded MN arrays ex vivo using full thickness porcine skin To investigate insertion properties of RALA/pCMV-Luc nanoparticleloaded PVP MNs, penetration studies were carried out using fullthickness neonatal porcine skin, previously determined to be a good model for human skin in terms of hair density and physical characteristics [23]. Prior to performing insertion studies, skin was shaved using a disposable razor and placed onto a sheet of dental wax (Anutex®, Kemdent Works, UK) for support with the SC facing towards the environment. MN arrays were inserted into the skin using spring-loaded applicators pre-set at forces of 8, 11 and 16 N, applied 3 times to the base of the MN array. Additionally, manual insertion efficacy was assessed by application of gentle thumb pressure to the base of the MN array for 30 s. The skin was immediately viewed using the VivoSight® EX1301 Optical Coherence Tomography microscope (OCT) (Michelson Diagnostics Ltd., UK) and images analysed using Image J software to determine penetration depth of the MNs.

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2.2.17. In vivo delivery of RALA/pCMV-Luc nanoparticles via intradermal injection pCMV-Luc or RALA/pCMV-Luc nanoparticles N:P ratio 10 containing 20 μg DNA (100 μl) were intradermally injected with a hypodermic needle into the rear dorsum of C57BL/6 mice, 3 mice per group. Mice were analysed via whole body imaging using an In Vivo Imaging System (IVIS) (PerkinElmer, UK) at 24 h and 48 h post administration, followed by sacrifice and harvesting of organs for ex vivo IVIS analysis. 2.2.18. In vivo dissolution of RALA/pCMV-Luc nanoparticle-loaded PVP MN arrays The dissolution rate of RALA/pCMV-Luc nanoparticle-loaded PVP MN arrays was determined in vivo to confirm the MN arrays are sufficiently robust to penetrate the SC and have appropriate characteristics for dissolution in situ. Nanoparticle-loaded MN arrays were applied to the dorsal ear skin of C57BL/6 mice by application of gentle manual pressure to the baseplate of the MN array for 5 min. OCT images were obtained immediately prior to MN insertion, 1, 5 and 15 min during MN dissolution and also post-insertion, when the MN array had been removed from the skin. Images were analysed using Image J software. 2.2.19. In vivo MN delivery and microscopic analysis of fluorescently labelled RALA/pCMV-Luc nanoparticles pCMV-Luc was fluorescently labelled with Cy-3 using a MirusBio LabelIt nucleic acid labelling kit (Cambridge Bioscience, Cambridge, UK) according to manufacturer's instructions. RALA/Cy-3-pCMV-Luc nanoparticles N:P ratio 10 were formulated into PVP MNs as described in Section 2.2.11. MN arrays were applied to the dorsal ear skin of C57BL/6 mice by application of gentle manual pressure to the baseplate of the MN array for 5 min. 1 h post application mice were euthanized, ear tissue was removed and fixed in 4% formaldehyde (Sigma-Aldrich, UK). Tissue was mounted onto microscope slide (VWR, UK) using 100% glycerol (Sigma-Aldrich, UK) and imaged using a TSC SP5-Leica Microsystems confocal microscope (Leica, UK) and analysed using LAS AF Lite software (Leica, UK). 2.2.20. In vivo delivery of RALA/pCMV-Luc nanoparticles via dissolving MN array MN arrays loaded with RALA/pCMV-Luc nanoparticles N:P ratio 10 were applied to the dorsal ear skin of mice, one per ear, as described in Section 2.2.19. At 6, 24 and 48 h post application, mice were analysed for bioluminescence via whole body imaging. Mice were intraperitoneally administered 200 μl D-luciferin K salt (15 mg/ml) (PerkinElmer, UK), and following 10 min incubation, were analysed for bioluminescence using a Xenogen IVIS 200 Imaging System (PerkinElmer, UK). Mice were anesthetised using isoflurane, and imaged using a three min exposure. 2.2.21. Bioluminescence imaging of luciferase expression and biodistribution ex vivo Following in vivo imaging, mice were sacrificed by cervical dislocation and the heart, lungs, liver, kidneys and spleen were harvested. The organs were bathed in 15 mg/ml D-Luciferin potassium salt in PBS for 10 min and were imaged as above. The bioluminescence of individual organs were determined using Living Image® 3.2 software (Leica, UK). 2.3. Statistical analysis Unless otherwise stated, three independent experiments were carried out for each analysis and the results are presented as mean ± SEM (n = 3). Statistically significant differences were calculated using the one-tailed unpaired t-test or one way analysis of variance with a p value of ≤ 0.05 considered significant. Statistical analyses were performed using Prism 5.0 (GraphPad Software, CA).

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3. Results and discussion 3.1. Characterisation and stability of RALA/pCMV-Luc nanoparticles Following formulation of RALA/pCMV-Luc nanoparticles, analysis of particle size and surface zeta potential was performed as it is known these physical parameters play a crucial role in cellular uptake [24]. Results indicate that the RALA peptide condenses the pCMV-Luc construct into nanoparticles b100 nm from N:P 6 upwards; the nanoparticles have a positive surface zeta potential, which increases as the N:P ratio (and consequently, the amount of cationic RALA) in the formulation increases. RALA/pCMV-Luc nanoparticles, N:P ratio 10, were then subjected to a series of studies designed to investigate stability under a number of adverse conditions, including exposure to serum, prolonged storage and elevated temperatures. DNA integrity was compromised by incubation in serum for as little as two hours, as evidenced by the smeared pattern of DNA following electrophoresis (Fig. 1Bi); conversely, the integrity of DNA that was complexed with RALA was unaffected by incubation in serum for up to 24 h (Fig. 1Bii). This demonstrates that the DNA encapsulated within the nanoparticles was not adversely affected or degraded by the presence of serum proteins and enzymes, unlike the ‘naked’ DNA. Thus, RALA offers protection to the DNA against degradation in serum, a quality that is critical for nucleic acid delivery vehicles in vivo. Prolonged storage, up to 28 days, of the RALA/pCMV-Luc nanoparticles at room temperature did not adversely affect the physical properties of the nanoparticles, as illustrated in Fig. 1C. The nanoparticles consistently measured b100 nm and have a zeta potential of 10– 30 mV, demonstrating the nanoparticles remain complexed and retain their electrostatic interaction, preventing aggregation. Additionally, exposing RALA/pCMV-Luc nanoparticles to elevated temperatures, up to 40 °C, did not compromise the integrity of the nanoparticles as indicated in Fig. 1D, with particles consistently remaining b 100 nm in size. This inherent stability could circumvent the need for complex storage conditions commonly required for biopharmaceutical products such as protein vaccines and hormone therapies, an important consideration for the development of biopharmaceuticals, particularly for agents to be used clinically in countries with extreme climates [5]. Although pharmaceutical degradation is dependent on a number of factors, such as storage conditions, product stability and packaging, it is thought the most important factor in degradation is temperature [25].

3.2. Compatibility of RALA/pDNA nanoparticles in PVP matrix Previous studies incorporating pDNA into MN coatings and polymeric matrices such as carboxymethylcellulose and poly(lactic-co-glycolic acid) have demonstrated that the dissolution of the polymer can adversely affect the stability and structure of the pDNA [26,7,27]. Thus it was necessary to determine if the polymer PVP matrix adversely affects the pDNA ± RALA. The stability of pCMV-Luc ± RALA within the PVP matrix was evaluated 48 h post incorporation into the polymer to determine if the integrity of either the DNA or the nanoparticles had been compromised. As illustrated in Fig. 2A, pCMV-Luc was released from the PVP upon dissolution as indicated by migration through the agarose gel (Lane 6). Comparison of the bands produced by this released DNA to those of unencapsulated stock DNA (Lane 1) indicate no degradation of the DNA had occurred upon incorporation into the PVP. Furthermore, RALA/pCMV-Luc nanoparticles incorporated into the polymer remained intact, illustrated in Lane 7, by the inability of the DNA to migrate down the agarose gel (i.e. retention of nanoparticles in the well). However, upon addition of a decomplexing agent, 10% SDS, to these formulations, DNA was released from the nanoparticles and migrated down the gel indicating once again that DNA was not degraded in the formulation and could be released from the polymeric matrix.

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Fig. 1. Characterisation and stability of RALA/pCMV-Luc nanoparticles. (A) Particle size and zeta potential of RALA/pCMV-Luc nanoparticles N:P ratios 4, 6, 8, 10 and 12; (B) Serum stability of (i) pCMV-Luc and (ii) RALA/pCMV-Luc nanoparticles N:P ratio 10 incubated for up to 24 h in serum at 37 °C. Following incubation, detergent SDS was added to decomplex the nanoparticles to verify integrity of DNA; (C) Particle size and zeta potential of RALA/pCMV-Luc nanoparticles N:P ratio 10 incubated at room temperature for up to 28 days; (D) Particle size analysis of RALA/pCMV-Luc nanoparticles N:P ratio 10 incubated between 4 and 40 °C. (N = 3, mean ± SEM).

To determine the biocompatibility of the PVP formulation, in vitro studies of its cytotoxicity were performed. Fibroblast cells were used as a model cell lineas an abundant cell type within the skin. NCTC-929

fibroblast cells were exposed to a range of concentrations of PVP for 24 h. The concentrations of PVP used in this study exceeded those expected to be deposited upon MN dissolution in the skin in vivo.

Fig. 2. Compatibility of RALA/pDNA nanoparticles with PVP matrix. (A) Agarose gel analysis of pCMV-Luc and RALA/pCMV-Luc release from PVP matrix; (B) NCTC-929 fibroblast cell viability following exposure to varying concentrations of PVP in vitro for 24 h; (C) Fluorescent microscope images of NCTC-929 cells 48 h post transfection with pEGFP-N1 ± RALA released from PVP matrix; (D) Flow cytometric analysis of GFP expression 48 h following transfection with pEGFP-N1 ± RALA released from PVP matrix. (N = 3, mean ± SEM). (n.s. = no statistically significant difference).

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Calculated by volume, the protruding MN tips on each array will be composed of 5.1 mg of 20% PVP, which will be deposited over a 1 cm3 area. Following exposure to 5 and 10 mg/ml the percentage cell viability was 98.3% and 94.1% respectively (p = 0.398 and 0.0616). Thus, at 5 and 10 mg/ml concentrations there was no statistically significant decrease in cell viability suggesting that the PVP formulation used in the MN fabrication in this study will not result in significant cytotoxicity. The functionality of pEGFP-N1 ± RALA following release from the PVP matrix was also investigated. The nanoparticle-polymer formulations were dissolved and used to transfect NCTC-929 fibroblast cells. The degree of fluorescence produced by cells transfected with pEGFPN1 released from PVP was not appreciably different from untransfected cells, whereas the degree of fluorescence produced when cells were transfected with nanoparticles released from PVP was significantly higher, and correlated with increasing N:P ratio (up to N:P 6) (Fig. 2C & D). Thus, these results confirm the necessity of the RALA peptide to enhance transfection efficacy [18]. These results also confirm that incorporation into, and release from the PVP formulation does not compromise functionality of the RALA/pEGFP-N1 nanoparticles. 3.3. Evaluation of RALA/pCMV-Luc nanoparticle-loaded polymeric MN arrays Following determination that the incorporation of RALA/pDNA nanoparticles into the PVP matrix does not adversely affect the functionality of the nanoparticles, it was necessary to determine if the nanoparticle-PVP formulation can be used to manufacture MN arrays fit for purpose. Nanoparticle-loaded MN arrays, containing 36 μg of

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DNA, were manufactured as described previously and imaged using SEM. Fig. 3A shows that MNs manufactured using this 2-step centrifugation process produce uniform MN arrays with needles of consistent length and width that are exact replicates of the mould used to fabricate them. However, previous studies involving the development of dissolving MN arrays have reported the incorporation of cargo into the MNs can compromise the physical characteristics of the MN array [28,29]. Thus, to determine if the inclusion of the RALA/pDNA nanoparticles into the PVP formulation compromised the physical integrity of the polymeric MN arrays, a texture analyser was used to apply a constant force of 45 N per array, and the reduction in height between the various MN formulations compared. Following compression of all three formulations, the percentage height reduction was approx. 11%. Thus, inclusion of nanoparticles in the formulation did not significantly impact on the mechanical strength of the polymeric MNs (Fig. 3B). When manufacturing the cargo-loaded MN arrays, 36 μg DNA was incorporated into the formulation, however, not all of this DNA will be present in the final MN array. It is possible to see in Fig. 3C that approx. 17.2 ± 1.6 μg and 21 ± 3.2 μg of DNA were present in the MN arrays loaded with pCMV-Luc and RALA/pCMV-Luc nanoparticles respectively. A proportion of the loaded DNA was lost by the ‘side-wall’ removal of the array, 6.6 μg and 6 μg for the pCMV-Luc and RALA/pCMV-Luc formulations respectively. The sum of the quantification of the side-walls and MN arrays do not equal 36 μg for either formulation, suggesting a proportion of the cargo is lost during the manufacturing process. Therefore manufacturing and scale up would need to be further optimised before this MN technology could progress towards clinical application. One reported method used to minimise cargo loss is the removal of PVP-cargo

Fig. 3. Evaluation of RALA/pCMV-Luc nanoparticle-loaded PVP MN arrays. (A) SEM image of RALA/pCMV-Luc nanoparticle-loaded PVP MN array; (B) Percentage height reduction of PVP MN arrays with no cargo, pCMV-Luc and RALA/pCMV-Luc nanoparticles; (C) Quantification of DNA content in DNA and nanoparticle-loaded PVP MN arrays; (D) (i) In vitro cumulative release profile of DNA released from nanoparticle-loaded PVP MN arrays across neonatal porcine skin up to 24 h; (ii) Percentage DNA release from nanoparticle-loaded PVP MN arrays across neonatal porcine skin; (E) Percentage penetration NP-loaded PVP MNs inserted into full-thickness porcine skin following application of varying forces. (N = 3, mean ± SEM).

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excess from the mould once the MN cavities have been filled. This excess can be returned to the master mix and subsequently reused, however, this system may not be applicable on a large-scale [30]. Franz-cell diffusion studies were carried out to investigate the release profile of RALA/pCMV-Luc nanoparticles from dissolving PVP MN arrays across dermatomed neonatal porcine skin (300 μm), a commonly used skin model for such studies [22]. As illustrated in Fig. 3D, pCMVLuc was detected in the receiver compartment of the Franz diffusion cell within 1 h following application of the MN array. The DNA concentration in the receiver compartment of the apparatus increased in a time-dependent manner until termination of the experiment at 24 h, at which point the MNs had fully dissolved. The dissolving MN arrays delivered nanoparticles containing approx. 23 ± 3.6 μg DNA across the porcine skin, meaning approx. 64% of the initially loaded cargo was delivered across the porcine skin model. However, as discussed previously, a proportion of the loaded nanoparticles are lost during manufacture suggesting that the nanoparticle delivery from this dissolving formulation is efficient, releasing approx. 100% of the nanoparticle cargo present in the MN array across the neonatal porcine skin during these diffusion studies. There are limitations to diffusion studies using model membranes such as dermatomed porcine skin in that the MN length is greater than the thickness of the skin, thus, a proportion of the cargo-loaded MN tips will be in contact with the receiver medium, potentially accelerating dissolution. Additionally, the skin cannot ‘recover’ and close the pores through the SC created by the MNs, as would happen within an in vivo setting using viable skin. The combination of these limitations may result in an overestimation of cargo delivery [22]. Having ascertained that the RALA/DNA NPs can be incorporated into and subsequently released from dissolving PVP MN arrays without decomplexation and loss of functionality it was necessary to determine if the MNs were of sufficient strength to penetrate full thickness

neonatal porcine skin, a common model for human skin [22,23] and also, what force was required for efficient breach of the SC and penetration into the epidermis. Nanoparticle-loaded MN arrays were prepared, applied to skin using 8, 11 and 16 N forces and also, manual pressure for comparison of the efficacy of penetration into the skin. Fig. 3E illustrates the percentage needle penetration into the skin as determined by OCT imaging. Of the range of forces employed in this study manual pressure resulted in most effective needle penetration with 84.3% of the MN length breaching the SC. It has been previously reported that penetration depths correlate with the forces applied during insertion within the range of 0–50 N, with manual application resulting in an average force of 20 N [31]. These results suggest that application of manual pressure to the MN array is an appropriate force and method of application to the skin in comparison to the use of an applicator device applying pre-determined forces. Additionally, as MN devices move from development-phase to clinically applicable low-cost products, patient acceptability and the usability of the devices are important factors, however, manual patient self-application of MN arrays has been successfully evaluated previously by utilising a pressure responsive backing on the MN patch [32]. 3.4. Stability of RALA/pCMV-Luc nanoparticle-loaded PVP MN arrays under prolonged and adverse conditions To evaluate the storage stability of RALA/pCMV-Luc-loaded PVP MN arrays, samples were stored for up to 28 days in temperature controlled environments at 4, 20 and 37 °C. Agarose gel analysis of RALA/pCMVLuc nanoparticles released from the PVP matrix following storage at room temperature revealed that the nanoparticles were still intact following incubation for 0, 7, 14, 21 and 28 days as indicated by an absence of DNA migration through the gel in the lanes labelled ‘Nanoparticles’ in Fig. 4A. Furthermore, when SDS was added to lyse the nanoparticle

Fig. 4. Stability of RALA/pCMV-Luc nanoparticle-loaded PVP MN arrays under prolonged and adverse conditions. (A) Agarose gel analysis of pCMV-Luc and RALA/pCMV-Luc release from PVP matrix following storage at room temperature for up to 28 days; (B) Percentage height reduction of PVP MN arrays with no cargo, pCMV-Luc and RALA/pCMV-Luc nanoparticles following storage at 4, 20 and 37 °C for 28 days; (C) Fluorescent microscope images of NCTC-929 cells 48 h post transfection with pEGFP-N1 ± RALA from PVP matrix following storage at (i) 4 °C, (ii) 20 °C and (iii) 37 °C; (D) Flow cytometric analysis of GFP expression 48 h following transfection with pEGFP-N1 ± RALA from PVP matrix following storage at 4, 20 and 37 °C for 28 days. (N = 3, mean ± SEM).

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formation, DNA did migrate down the gel and the bands produced were comparable to those in the ‘pCMV-Luc’ control lane, indicating that there was no alteration in the DNA conformation following these incubations. Compression studies carried out on the MN arrays revealed that they preserved their mechanical strength during storage, as illustrated in Fig. 4B. Based on the percentage height reduction there was no statistically significant difference between the strength of the needles regardless of cargo or storage conditions, suggesting that the MN strength for all formulations investigated was maintained under the various storage conditions for up to 28 days. The functionality of the RALA/pDNA nanoparticles was also confirmed by transfection studies as illustrated in Fig. 4C and D. Following storage for 28 days at 4, 20 and 37 °C, transfection efficacy for the nanoparticle groups was approx. 15%, consistent with the transfection efficacy described in Fig. 2D. Thus, the nanoparticles are not compromised following prolonged storage under these conditions and maintain functionality. 3.5. In vivo and ex vivo analysis of RALA/pCMV-Luc nanoparticle-loaded MN efficacy To determine the efficacy, timing and distribution of protein expression following intradermal delivery, pCMV-Luc and RALA/pCMV-Luc

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nanoparticles were injected into the dorsum of C57BL/6 mice using a hypodermic needle; 20 μg pDNA per injection was administered and luciferase expression analysed at 24 and 48 h post administration. It is evident in Fig. 5A that the levels of luciferase expression are superior following administration of the nanoparticle formulation compared to naked pDNA at both time points investigated. Furthermore, this study validates that nanoparticle delivery into the skin can result in prolonged gene expression at distal sites thus, they may be delivered via this route for the purposes of systemic gene therapy. Thus, the nanoparticle formulations were further investigated in vivo. To confirm if the nanoparticle-loaded dissolving MNs can be successfully used for intradermal delivery of the in vivo, OCT was employed to allow non-invasive visualisation of MN dissolution while inserted in murine skin [33]. We evaluated the dissolution profile of the nanoparticle-loaded MNs as illustrated in Fig. 5B and found that the greatest dissolution of the MNs in the skin occurred in the first 5 min, MNs had dissolved and lost their needle profile, continuing to dissolve up to 15 min. This rapid dissolution of the PVP MNs is similar to that reported by Guo et al. (2013) and Qiu et al. (2015) who observed dissolution of PVP MN tips in porcine ears and mouse skin respectively, within 3 min [30,34]. We further confirmed in situ cargo delivery through analysis of the distribution of fluorescently labelled RALA/Cy3-pCMV-Luc

Fig. 5. In vivo and ex vivo analysis of RALA/pCMV-Luc nanoparticle-loaded MN efficacy. (A) Representative images of luciferase gene expression following intradermal injection of pCMVLuc and RALA/pCMV-Luc nanoparticles; (B) OCT analysis of MN dissolution over time in mouse ear in vivo; (C) Confocal microscopic images of mouse ear 1 h following application of ‘Empty’ MN array and MN array containing Cy3 fluorescently labelled RALA/pCMV-Luc nanoparticles. (D) Representative (i) Dorsal; (ii) Harvested organ images captured via IVIS analysis of biodistribution of gene expression 48 h following MN delivery of RALA/pCMV-Luc NPs; (E) Quantification of bioluminescence detected in specific organs 6, 24 and 48 h post MN delivery of RALA/pCMV-Luc nanoparticles. (N = 3, mean ± SEM).

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nanoparticles in vivo 1 h post MN application (Fig. 5C). These studies confirm rapid dissolution of the dissolving PVP MN array formulation and delivery of the macromolecular cargo within the skin layers, similar to that reported by Ke et al. (2012) for the delivery of PLGA microspheres [35] and Guo et al. (2013) for the delivery of antigen-loaded liposomes [30]. Finally, evaluation of the efficacy of the nanoparticle-MN platform for eliciting gene expression in vivo was determined. It is thought that only macromolecular cargo present within the needles of the array will be deposited into the skin due to pore closure following removal of the MN array, as illustrated in Fig. 5B ‘post insertion’ image. Approx. 7.8 μg DNA is present within the needles of each array based on the volume of the needle cavities, thus approx. 15.6 μg DNA was delivered per treatment i.e. a MN array applied to both ears per animal. Luciferase expression was analysed at 6, 24 and 48 h post MN application. Although no bioluminescence was detected in whole body imaging analysis (Fig. 5D), luciferase expression was detectable in the livers and kidneys of mice 6 h post administration of MNs, and was detectable up to 48 h post-administration (Fig. 5E). The inability to accurately determine the quantity of DNA delivered from the dissolving MN patch into the skin, not that deposited onto the surface of the site of application, is a limiting factor in the progression of this technology which must be addressed prior to use in a clinical setting [13]. This study confirms that the delivery of RALA/pDNA nanoparticles via the dissolving MN platform developed in this study is capable of eliciting systemic protein expression in vivo. This biodistribution of protein expression in the liver and kidneys is indicative of the nanoparticles entering the systemic circulation following MN dissolution, followed by dissemination and retention in highly vascularised regions. In this respect, our current observations are comparable to those reported in our previous study [18], when RALA/pLuc nanoparticles were delivered intravenously, and to MPG-8 peptide and calcium phosphate nanoparticles [36,37]. It has been hypothesised in the literature that nanoparticle aggregates in organs such as the liver are ineffective gene delivery agents [38]. However, in this research the luciferase expression in the liver and kidneys was either maintained or elevated at 48 h post administration compared to 6 and 24 h, suggesting that the nanoparticles have retained their functionality and have not been cleared by these excretory organs. Thus, the natural biodistribution profile of this delivery platform could be exploited for delivery of gene therapy to the liver and/or kidneys without having to employ a targeting element to the system. 4. Conclusion In the present study, we have for the first time designed, developed and evaluated a novel two-tier gene delivery system based on the combination of our original cationic peptide gene delivery vehicle to overcome the intracellular barriers to gene therapy and a dissolving MN delivery platform as an alternative to conventional parenteral delivery. The work presented here illustrates the potential of dissolving MNs to deliver peptide/DNA nanoparticle cargo at doses capable of generating detectible levels of reporter gene expression in vivo. With further refinement this ‘smart-system’ technology platform could be an exciting nucleic acid delivery device for a number of pathologies. Acknowledgements This study was supported by a research grant from the Royal Society (RG2010R2). References [1] S.A. Coulman, A. Anstey, C. Gateley, A. Morrissey, P. McLoughlin, C. Allender, J.C. Birchall, Microneedle mediated delivery of nanoparticles into human skin, Int. J. Pharm. 366 (Jan 21, 2009) 190–200, http://dx.doi.org/10.1016/j.ijpharm.2008.08. 040.

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