NANO-00981; No of Pages 9

Nanomedicine: Nanotechnology, Biology, and Medicine xx (2014) xxx – xxx nanomedjournal.com

Triggered-release nanocapsules for drug delivery to the lungs

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Institute of Pharmaceutical Science, King's College London, London, UK Received 25 April 2014; accepted 24 July 2014

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Jasminder Chana, PhD, Ben Forbes, PhD, Stuart A. Jones, PhD⁎

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Abstract

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This study demonstrated the feasibility of trigger-responsive inhaled delivery of medicines using soft solid shelled nanocapsules. The delivery system was a 50 nm sized lipid rich capsule carrier that distended rapidly when mixed with an exogenous non-ionic surfactant trigger, Pluronic® L62D. Capsule distension was accompanied by solid shell permeabilisation which resulted in payload release from the carrier; 63.9 ± 16.3% within 1 h. In electrolyte rich aqueous fluids Pluronic® L62D was loosely aggregated, which we suggest to be the cause of its potency in lipid nanocapsule permeabilisation compared to other structurally similar molecules. The specificity of the interaction between L62D and the nanocapsule resulted in carrier payload delivery into human epithelial cells without any adverse effects on metabolic activity or barrier function. This effective, biocompatible, trigger-responsive delivery system provides a versatile platform technology for inhaled medicines. © 2014 Published by Elsevier Inc.

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Key words: Nanomedicines; Lipid nanocapsules; Permeabilisation; Pluronic® surfactant; Pulmonary drug delivery

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The rapid clearance of therapeutic agents from the lungs following inhalation presents a major challenge to treating pulmonary disorders such as asthma and chronic obstructive pulmonary disease using conventional respiratory medicines. 1 Nanocarriers provide a potential solution to this problem as their small size enhances their ability to moderate active molecule presentation to the tissue whilst limiting rapid clearance from the lung that occurs for larger particulate carriers. 2 -5 However, nanosized drug carrier systems can suffer premature payload leakage upon storage and this renders these systems unsuitable for inhaled delivery and therefore new approaches are required for this route of administration. 6 Trigger-responsive delivery systems have the capability of switching between two states; one in which the drug is held within a carrier without significant release, termed ‘off’, and a second where drug is released, termed ‘on’. 7 They are typically used for ‘site-specific’ drug delivery once an active pharmaceutical compound has entered the body, but the concept has not as yet been adapted to meet the specific demands of inhaled

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Introduction

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medicines. In the lung, endogenous triggering, which in other areas of the body has been shown to be an effective way of targeting therapeutic agents, 8 is restricted by the need to exploit a physiological environment capable of inducing a unique chemical or physical change in the nanocarrier to initiate the delivery process. 9 However, the characteristics of some reported exogenously triggered systems appear well matched to inhaled drug administration. 10,11 Portability and ease of use are important considerations for patients who inhale medicines, thus exogenous triggered delivery systems that do not use specialist equipment for administration, e.g. systems requiring co-administration of chemical triggers, offer the most convenient approach. 12-18 Pulmonary administration devices are available that allow two liquids to be combined at the point of administration, 19 providing a system where the trigger and the nanomedicine mix at the mucosal surface of the lung where the aerosol deposits. For such a system to be effective, however, the carrier must possess sufficient responsiveness to the trigger to enable control of drug release. 20-23 This aspect of chemicallytriggered nanomedicines appears to be more problematic than codelivery to the mucosal surface because many highly reactive

This work was funded by the BBSRC. ⁎Corresponding author at: Institute of Pharmaceutical Science, King's College London, 150 Stamford Street, London, UK. E-mail address: [email protected] (S.A. Jones). http://dx.doi.org/10.1016/j.nano.2014.07.012 1549-9634/© 2014 Published by Elsevier Inc. Please cite this article as: Chana J., et al., Triggered-release nanocapsules for drug delivery to the lungs. Nanomedicine: NBM 2014;xx:0-9, http://dx.doi. org/10.1016/j.nano.2014.07.012

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nano-sized carriers with the ability to release payloads have a tendency to induce potentially detrimental biological effects, e.g., disruption of cell homeostasis, 24 induction of inflammatory responses, 5 damage to cell membranes 25 or the induction of protein adsorption. 26 One trigger-responsive carrier system that appears distinct in its biocompatibility profile is the liposome. 27 Liposomal carriers have been reported to show limited immunogenic potential 28 and have the ability to respond to surfactant triggers, 10 but they are prone to drug leakage upon storage. 25,29 Nanocapsules formed by an emulsion phase inversion-precipitation technique exhibit similar properties to liposomes, but possess a solid outer shell that is less permeable. 30 The aim of this study was to investigate whether solid shelled lipid nanocapsules, fabricated using an Epikuron® Solutol® mixture (Figure 1), could be formed into a trigger responsive nanomedicine suitable for inhaled delivery. These charge neutral nanocapsules have recently been shown to be physically stable in protein-containing physiological salt solutions and they do not induce an inflammatory response in the lungs of mice. 31 Following on from the previous work, this study focused on the ability of a Pluronic triggering system to induce structural changes in the nanocapsules at an airway epithelial surface, the transfer of the released agent across the epithelial barrier and the response of the epithelial cells to the delivery system. The particles were characterised using photon correlation spectroscopy, Fourier transform infrared spectroscopy, transmission electron microscopy, x-ray spectroscopy and the

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Figure 1. Schematic of engineered lipid nanocapsules (LN) formed via emulsion phase inversion precipitation showing the shell structure and the proposed means by which the Pluronic® L62D trigger could insert and permeabilise the nanocapsule shell to provide trigger responsive delivery (nanocapsule structure taken from Heurtault et al 30).

delivery of rhodamine, used as a model drug, was determined using the MucilAir® system (constructed from primary human airway epithelial cells). The specificity of Pluronic® L62D to induce payload release was characterised by comparison with three structurally similar molecules.

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Materials and methods

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Materials

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Medium chain triglycerides (Labrafac® lipophile 1349), hydrogenated soybean lecithin (Epikuron® 200) and macrogol 15 hydroxystearate (Solutol® HS15) were kindly supplied by Gattefossé S.A. (Saint-Preist, France), Cargill GmbH (Germany) and BASF (Ludwigshafen, Germany), respectively. Rhodamine 6G was purchased from Sigma Aldrich, (Gillingham, UK). High performance liquid chromatography (HPLC) grade water was obtained from Fischer Scientific (Leicestershire, UK) and ethanol absolute from VWR (Leicestershire, UK). Pluronic® surfactants L62D, L44NF, L64 and L81 were sourced from BASF (New Jersey, USA). Cell culture reagents were sourced through Sigma Aldrich (Dorset, UK). Cell culture flasks (75 cm 2 with ventilated caps) and Transwell cell culture systems (0.33 cm 2 polyester, 0.4 μm pore size) were from Costar (through Fisher Scientific, Leicestershire, UK).

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Nanocapsule size was measured using a 1 in 20 dilution (v/v in HPLC grade water) of the original nanoparticle suspension, to ensure the viscosity of the sample was similar to that of pure water, with a Zetasier Nanoseries (Malvern Instruments, UK) at a scattering angle of 173°, 37 °C. Mean diameters were obtained from the normalised intensity distribution. The changes in the size of nanocapsules in response to Pluronic® surfactants were measured after suspensions at 37 °C were exposed to surfactant at concentrations of 50, 75, 100 and 150 mg per mL (n = 3) equivalent to nanocapsule:pluronic mass ratios of 1:0.25, 1:0.375, 1:0.5 and 1:0.75 (mass ratios calculated after particle purification). The formation of surfactant micelles by Pluronic® at the concentrations used in these studies was analysed in equivalent nanocapsule-free systems using the same sizing technique with and without NaCl (0.9% w/v).

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Transmission electron microscopy

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Nanocapsule suspensions were analysed using transmission electron microscopy (TEM) before and after exposure to Pluronic® L62D surfactant (150 mg/mL, 30 min post exposure). The suspension (3 μl) was applied to a Pioloform-coated copper grid with 1% w/v uranyl acetate and examined with a Tecnai T12 electron microscope (FEI, Oregon, USA). X-ray microanalysis was performed on nanocapsule suspensions in the absence and presence of Pluronic® L62D surfactant (150 mg/mL, 30 min) using the methods described above. High angle annular dark field (HAADF) scanning transmission electron microscopy (STEM) images were collected using a T20 (FEI, Oregon, USA) electron microscope fitted with a LaB6 emitter. X-ray microanalysis was carried out using the embedded TIA (Tecnai Imaging and Analysis) software with a super ultra thin (SUTW) detector (EDAX, Mahwah, USA).

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Fourier transform infra-red spectroscopy

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Nanocapsule suspensions were analysed using a Spectrum One FTIR spectrometer (Perkin Elmer, Beaconsfield, UK) fitted with a DuraSamplIRII diamond attenuated total reflectance attachment (Smiths detection, Warrington, UK). Each sample was applied in its liquid state to the diamond and 32 scans were performed, the solvent signal was subtracted from the trace using Spectrum One software (version 6). Spectra were

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Rhodamine loading and triggered release

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Rhodamine 6G was quantified using fluorescence spectroscopy (Perkin Elmer LS50B, Cambridgeshire, UK) in ethanol solutions using excitation and emission wavelengths of 534 and 552 nm respectively and slit widths of 2.5 nm (range 0.02-0.12 μg/mL, limit of detection 0.003 μg/mL, intraday coefficient of variation b 2%, no Pluronic® L62D matrix interference). Loading efficiency was determined using Amicon ultra 0.5 centrifugal filter devices with Ultracel 100 membranes (100 kDa molecular weight cut off; Millipore, UK) at 14,000 g for 40 min (Biofuge Pico centrifuge, Heraeus, Buckinghamshire, UK) with a full mass balance (rhodamine adheres to the filter apparatus). Rhodamine was extracted from the oil core of nanocapsules by exposure to ethanol. Rhodamine release from solid shell lipid nanocapsules (LN) using Pluronic® L62D as a trigger in an aqueous solution was tested at 37 °C with two different concentrations of trigger (50 mg/mL and 150 mg/mL, n = 3) using a 30 min exposure time and an identical rhodamine separation and quantification method as the loading studies.

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Calu-3 in situ release testing

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The Calu-3 human bronchial epithelial cell line, passages 30–40, were maintained in a humidified 5% CO 2 /95% atmospheric air incubator at 37 °C. Cell culture medium comprised 50:50 Dulbecco's Modified Eagle's Medium/Nutrient F-12 Ham's (500 mL) supplemented with foetal bovine serum (FBS, 50 mL), L -glutamine (200 mM, 5 ml) and gentamicin (0.5 mL). Cells were cultured in 75 cm 2 flasks with 20 mL medium. Medium was exchanged every 2-3 days and cells were passaged weekly at a 1:3 split ratio using 0.25% trypsin-EDTA solution. Cells were cultured at the air liquid interface on Transwell cell culture supports as described by Grainger et al 33 for the in situ release experiments. Cells were seeded at a density of 5 × 10 5 cells/cm 2 and introduced to the apical chamber of the Transwell cell culture support in 0.1 mL medium, with 0.5 mL medium in the basolateral chamber. After 2-3 days medium was aspirated from the apical and basolateral chambers and only replaced in the basolateral chamber. Medium was subsequently replaced every 2 days. Trans-epithelial electrical resistance (TER) of cell layers was measured using chopstick electrodes with an EVOM voltohmmeter (STX-2 and EVOM G, World Precision Instruments, Stevenage, UK) following the apical application of warm medium (100 μL, 37 °C) and 30 min equilibration time in an incubator. TER was calculated by subtracting the resistance of a cell-free culture

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Photon correlation spectroscopy (PCS)

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Nanocapsules were manufactured via precipitation from a stable emulsion following repeated phase inversion, as described previously. 29 Rhodamine, a model agent with intermediate lipophilicity (log P 1-4) 32 was incorporated into the nanocapsules by addition to the oil phase (50 μg per 1 g oil) as an acetonic solution (100 μg/mL). The nanocapsule suspensions were purified of excess excipients and larger particulate matter via centrifugation (Beckman L8-80 ultracentrifuge, Beckman Coulter, Buckinghamshire, UK) using 110,000 g at 25 °C for 1 h. The purified suspension layer was used for further experimental work and possessed a solid content of 206.7 ± 6.5 mg/mL, determined by recording the dry weight at equilibrium (n = 3).

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collected for the nanocapsules, phospholipid nanocapsule components and for the system following 0.25, 0.5. 1 and 1.5 h exposure to Pluronic® L62D at 150 mg/mL. In addition, spectra were obtained for nanocapsules after separation from the suspension using Amicon ultra 0.5 centrifugal filter devices with Ultracel 100 membranes (100 kDa molecular weight cut off; Millipore, UK). Samples were centrifuged for 40 min at 14,000 g at ambient temperature.

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Cell viability assay

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The MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was performed according to the methods of Grenha et al. 34 Calu-3 cells were seeded at a density of 1 × 10 4 cells/well in 96-well plates and cultured for 24 h in cell culture medium with a reduced FBS concentration of 2% v/v. Solutions of the surfactant in cell culture medium were prepared at a range of concentrations and equilibrated at 37 °C. Cells were either exposed to 100 μL of the surfactant or control solutions (cell culture medium as a positive control, 1% Triton X solution as a negative control) for 1 h, following which well contents were replaced with 200 μL cell culture medium and 50 μL of MTT in phosphate buffered saline (5 mg/mL). After 4 h incubation at 37 °C medium was removed from all the wells and replaced with 100 μL of SDS solution (10% w/v in 50:50 DMF:water) to solubilise any formazan crystals generated. Absorbance from each well was measured 16 h later using a

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Epithelium and culture medium was donated by Epithelix Sarl (Geneva, Switzerland) and was used as per manufacturer's instructions. The confluent cell layers were formed on identical Transwell supports to those used for Calu-3 cell culture. Cell layers were maintained in a humidified incubator as described for Calu-3 cell culture and the medium in the basolateral chamber was changed every 2-3 days. Cells were maintained for 3 weeks prior to use. TER was monitored as described for Calu-3 cell layers. Lipid nanocapsules applied to the MucilAir™ cell layers were suspended in Hank's balance salt

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MucilAir™ transport studies

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solution (HBSS, Sigma Aldrich) prior to use and the cell culture medium in the basolateral chamber was replaced with HBSS (0.6 mL) pre-warmed to 37 °C. Purified rhodamine loaded nanocapsule suspensions (100 μL) pre-warmed to 37 °C were administered to the apical compartment and the cell layers were returned to the incubator. After 6 h the apical chamber contents were carefully homogenised by repeat pipetting (5 operations) without touching the cell layer and then transferred to centrifugal filter devices. Apical samples were processed for 40 min at 14,000 g to separate nanocapsules from the continuous phase and then retained for assay. Fresh HBSS (100 μL) was then added to wash the cell layer to recover any remaining rhodamine in the apical compartment. This volume was homogenised using a pipette (5 operations) taking care not to disturb the cell layer and then removed for assay. Basolateral chamber contents were sampled and the cell layer was solubilised by applying 10% w/w SDS (200 μL) and leaving overnight in an incubator followed by dilution with ethanol. Calibration curves showed no matrix interference in HBSS or the SDS/ethanol mixture.

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insert (140 Ω) and correcting for the surface area of the Transwell cell culture support (0.3 cm 2). In situ release experiments were conducted 11-13 days post cell seeding on the Transwell inserts (selected for maximal TER and consistent cell layer properties 33). TER was recorded immediately before sample application, after 30 min incubation with sample, immediately before sample removal, and 24 h later, and reported as a percentage of the pre-experiment value. Test suspensions (250 μL) were prepared in cell culture medium prior to application to the cell layers and comprised nanocapsules (diluted 1 in 20 v/v, i.e. containing a solid mass of 10 mg/mL), Pluronic® L62D (2.5 mg/mL), nanocapsules and Pluronic® L62D (each at the same final concentration as tested individually, representing a mass ratio of 1:0.25) or a medium-only control (n = 3 per condition). Following 1 h incubation with the cells, apical chamber contents were homogenised and a 200 μL aliquot removed for assay. Cells were then washed twice with fresh cell culture medium to remove any residual sample traces and returned to the incubator. Apical chamber samples were prepared for assay using the centrifugal filter devices for 30 min as described previously. Rhodamine content of the nanocapsules was determined following dilution in ethanol (Cary Eclipse fluorescence spectrophotometer with microplate accessory, Varian, Victoria, Australia) using excitation and emission wavelengths of 534 and 552 nm respectively. Release was calculated from the percentage of rhodamine remaining in nanocapsules compared to that determined during loading.

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Figure 2. Nanocapsule swelling over time following exposure to Pluronic® L62D (left: black triangles), L44NF (grey diamonds), L64 (grey squares) at a nanocapsule:trigger mass ratio of 1:0.75, compared to a non-exposed nanocapsule control (unfilled circles). L81 was not soluble in the assay conditions. Data represent mean ± SD, n = 3. Properties of Pluronic® surfactants (right): molecular weight (MW); propylene oxide units (PO, y); ethylene oxide units (EO, x) and critical micelle concentration (CMC). 35

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Data were analysed using Statistical Package for Social Sciences (SPSS) software. One way analysis of variance (ANOVA) tests were performed to detect significant differences in data with a probability limit of 0.05. Pairwise comparisons within each data set were performed with post hoc Tukey tests. The probability limit was set at 0.05.

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Results

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Pluronic®-induced nanocapsule swelling

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Lipid nanocapsules were fabricated using an emulsion phase inversion-precipitation method for use in subsequent triggering studies. The resultant nanocapsules possessed a unimodal, normally distributed population with a mean diameter of 50 ± 1 nm and a polydispersity index of 0.04 ± 0.01 (n = 3) when characterised using PCS. The solid content of the nanocapsule suspension was high at 206.7 ± 6.5 mg/mL (n = 3). PCS analysis performed on the Pluronic® surfactant solutions at the concentrations used in the triggering studies revealed the presence of a multimodal size distribution with peaks at circa 2 nm, 20 nm and 200 nm (supplementary material, Figure S1). However, the introduction of nanocapsules into the surfactant solutions resulted in almost complete disappearance of surfactant peaks due to the 202000 fold greater light scattering signal of the nanocapsules compared to surfactant (data not shown), which made the nanocapsules easy to distinguish in the sizing data analysis. Nanocapsule suspensions were incubated with Pluronic® surfactants at a mass ratio of 1:0.75 (1 mL suspension:150 mg Pluronic®) and nanocapsule size was monitored via PCS over 48 h. Exposure to Pluronic® L62D induced a substantial increase in nanocapsule size over time, resulting in a mean size of 234 ± 5 nm at 24 h (Figure 2). A more modest swelling response of 135 ± 13 nm at 24 h was detected when the nanocapsules were exposed to Pluronic® L44NF. No significant change in size compared to control (51 ± 1 nm) was observed for Pluronic® L64. Pluronic® L81 was insoluble in the aqueous nano-suspension at this concentration. A secondary peak in the size intensity distribution emerged for nanocapsules exposed to the Pluronic® L62D at 48 h, corresponding to circa 20 nm (supplementary material, Figure S2).

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Nanocapsule shell permeabilisation

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The potential for Pluronic® surfactants to trigger nanocapsule permeabilisation was assessed via TEM and FTIR following their co-incubation at 37 °C (nanocapsule:L62D mass ratio 1:0.75). The TEM images of nanocapsules corroborated PCS sizing data in that non-triggered control nanocapsules were approximately 50 nm in size (Figure 3, A) and appeared to have

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Statistical analysis

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spectrophotometer (SpectraMax 190, Molecular Devices, USA) at 570 nm, corrected for background absorbance at 650 nm. Absorbance was used to determine cell viability by comparing to the negative and positive controls. 34 The assay was performed in triplicate, with three to six repeats for each Pluronic® concentration on each occasion.

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Figure 3. Transmission electron microscopy (TEM) images of lipid nanocapsule suspensions, (A) before exposure to Pluronic® L62D surfactant, and (B) 30 min after exposure to Pluronic® L62D. The X-ray microanalysis of nanocapsules exposed to Pluronic® L62D surfactant showing the elemental composition of (C) the nanocapsules core and (D) the sample grid. The uranium (U) was from the uranyl acetate stain, copper (Cu) and silica (Si) originated from the sample grid.

swollen to N 100 nm after exposure to Pluronic® L62D surfactant (Figure 3, B). The control nanocapsules appeared light against a dark background due to the inability of the uranyl stain to penetrate the capsule shell, but following exposure to Pluronic® the opposite staining pattern was observed. Uranium was at a significantly higher concentration within the nanocapsule core than the exterior following exposure to Pluronic® L62D (Figure 3, C and D), which was not the case for the nontriggered control nanocapsules (supplementary material, Figure S3). FTIR analyses of the nanocapsules indicated a shift in the frequency of the CH2 symmetric stretch from 2855 cm − 1 to 2858 cm − 1 when they were exposed to the Pluronic® L62D trigger (supplementary material, Figure S4).

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Triggered release

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Rhodamine loaded into the nanocapsules with a high encapsulation efficiency (82.1 ± 3.4%, n = 3). Co-incubation of the nanocapsules with the Pluronic® L62D trigger for 1 h (1 mL suspension: 150 mg L62D = mass ratio of 1:0.75) in the

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The swelling response of the nanocapsules was concentrationdependent for two of the Pluronic®, L62D and L44NF (Figure 5, A and B). Each incremental increase in concentration of L62D produced a significant growth in nanocapsule size, at both 6 and 24 h post-exposure (ANOVA, P b 0.05). A similar trend was observed for L44NF, but statistically significant differences in size with incremental increases in concentration were only seen at 24 h (ANOVA, P b 0.05). The response to Pluronic® L64 was not concentration dependent, with no significant increase in size with concentration up to 24 h (supplementary material, Figure S7).

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Discussion

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The size and polydispersity of the nanocapsules employed in this work were consistent with those previously reported using similar preparation methods. 21,29 When the Pluronic surfactants were added to the nanocapsules the light scattering peak associated with the aggregated surfactant in solution diminished (data not shown). This change suggested that the amphiphilic surfactant molecules are re-arranging in a manner that would facilitate adsorption at the solid/liquid interface of the nanocapsule. Langmuirian surface adsorption of Pluronic® surfactants onto latex particles has previously demonstrated that maximal adsorption occurs at concentrations just above the CMC and typically results in small size increase of 2-5 nm. 36,37 A significant increase in nanocapsule size followed the co-incubation of the particles and the surfactant that was of a magnitude that could not be accounted for simply by the adsorption of Pluronic® monomers onto the nanocapsule surface. Electron microscopy images visually illustrated the size increase and showed the associated uranyl acetate stain influx into the nanocapsule which suggested that Pluronic® adsorption was a precursor to nanocapsule distension and permeabilisation.

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Pluronic® trigger – nanocapsule specificity

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Exposure to Pluronic® L81 was only possible at 50 mg/mL (mass ratio of 1:0.25) due to poor solubility of the surfactant in the aqueous nanocapsule suspension, but at this concentration no significant difference was found in nanocapsule size compared to non-exposed control over a 24 h period (ANOVA, P N 0.05, supplementary material, Figure S8). In order to investigate the influence of surfactant aggregation behaviour on triggering nanocapsule distension the efficiency of the triggering process was determined as a function of electrolyte concentration. PCS studies of the surfactant solutions in the presence of NaCl revealed that L62D was most sensitive to changes in electrolyte content, as indicated by a decrease in the sample derived count rate when the solutions were diluted with water (Figure 6, A, associated intensity distributions in supplementary material, Figure S9). However, hyperosmolar concentrations were required to observe a corresponding effect on the 24 h nanocapsule distension response (Figure 6, B).

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absence of the cell released 13.0 ± 1.7% of the encapsulated rhodamine and 34.0 ± 18.2% after 5 h (Figure 4, A). The untriggered nanocapsules and those exposed to a mass ratio of 1:0.25 Pluronic® L62D (1 mL suspension: 50 mg L62D) released less than b 10% of the loaded rhodamine. At the surface of air-interfaced Calu-3 respiratory airway cell layers 2.5 mg/mL of Pluronic® L62D (equivalent mass ratio 1:0.25) released 63.9 ± 16.3% of rhodamine, compared to the untriggered control release of 25.0 ± 17.8% (ANOVA p b 0.05; see supplementary data; S5). At the surface of the MucilAir™ epithelial layers 19.7 ± 1.2% of the nanocapsule-loaded rhodamine was released by Pluronic® L62D vs 12.0 ± 0.4% without the trigger (ANOVA, P b 0.05) (Figure 4, B). After 6 h, 5.7 ± 0.2% of the administered rhodamine had transferred across the MucilAir™ epithelial layer compared to 4.1 ± 0.1% for the untriggered control (ANOVA, P b 0.05, Figure 4, B). The pre-experiment TER, a measure of cell layer barrier function, was 479 ± 199 Ω ∙ cm 2 for the Calu-3 cells and this remained unchanged during the triggering experiment and for 24 h afterwards. The TER of the MucilAir epithelium was 435 ± 11.8 Ω ∙ cm 2 at the start of the experiment and was recorded as 243.1 ± 18.1 at the 6 h time point. The MTT assay revealed no observable effect of the Pluronic® L62D trigger on Calu-3 cell metabolic activity for concentrations up to 5 mg/mL, and an IC50 of 12.48 ± 1.1 mg/mL (see supplementary data; S6) which was approximately five times greater than concentrations used in the in situ release experiments for this cell line. Interestingly, a concentration of 15 mg/mL was welltolerated by MucilAir cells (Figure 4, B).

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Figure 4. Rhodamine release from solid shell lipid nanocapsules (LN) using Pluronic® L62D as a trigger in an aqueous solution (A) and rhodamine disposition in the apical chamber, cell and basolateral chamber upon exposure of the MucilAir™ epithelium to the LN in the absence and presence of Pluronic® L62D (B). Data represent mean ± SD, n = 3.

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loss of the peak shoulder, similar to that reported when lipid membranes are fluidised, 39 occurred in a similar time-frame. The attenuated reflectance FTIR results indicated that the fluidity of hydrocarbon chains in the lipid nanocapsule shell increased, 40 -42 but due to the overlapping peaks in the spectra the technique could not specifically identify which component interacted with the surfactant. Despite this the FTIR results, in combination with the PCS and TEM data, provided a strong basis to support the hypothesis that L62D inserts into the lipid shell, increases the conformational freedom of the lipids and resulted in nanocapsule distension. Rhodamine release from the nanocapsules was rapid after trigger exposure and this aligned well with reported data on liposomal carriers. 38 The release of only 10% rhodamine in the absence of the trigger indicated that the nanocapsules remained intact in the absence of the trigger and was similar to that reported by Xiong and co-workers (2012), who formed a highly specific nanocapsule system for permeabilisation by lipase. 18 The triggered release of rhodamine operated effectively at the surface of confluent Calu-3 and MucilAir™ cell layers. Rhodamine accumulated in the cells, which restricted the transport of the molecule across the epithelial barrier, and this reduced the impact of the particle triggering when considering transepithelial transport. It would be interesting to compare in future work how controlled release from the lipid nanocapsules 43,44 influences the disposition of more hydrophilic inhaled molecules such as salbutamol or terbutaline. The barrier integrity and the viability of epithelial cell layers were typical of that reported in the literature 33 and they were shown to withstand the triggering process. Pluronic® L62D selectivity towards the

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For Pluronic® surfactants, increased PO block length and PO: EO ratio have been reported to increase surfactant hydrophobicity and the extent of lipid bilayer disruption. 25,30 For Pluronic® with intermediate PO block lengths (i.e. L44NF, L62D, L64) partial insertion into bilayers has been cited as the primary means of membrane interaction, with both EO segments residing on the same side of the membrane and the PO chain protruding into the hydrophobic domain. 30 The superiority of Pluronic® L62D in triggering nanocapsule swelling compared to L44NF and L64 was consistent with the partial insertion theory. A more disruptive effect on membrane integrity has been noted when surfactant molecules mimic the structure of the acyl chain region of the lipid membrane into which they are inserting, 30 indicating the importance of the 32 unit PO block of L62D. The observation that L44NF produced an enhanced distension response compared to L64 despite being less hydrophobic suggested that Pluronic® interaction with the nanocapsule shell was not the only factor driving the nanocapsule swelling, which was supported by further investigations into the surfactantnanocapsule interaction. The accumulation of the uranyl acetate stain into the nanocapsule cavity following exposure to Pluronic®, observed via electron microscopy, indicates that the nanocapsules were permeabilised within 30 min of the trigger exposure. Similar observations of vesicle deformation and permeabilisation by Pluronic® surfactants have been reported for more flexible phosphatidylcholine liposomes, 38 but this is thought to be the first demonstration of this effect for solid shell nanocapsules suitable for administration to the lung. 31 FTIR spectral data supported this conclusion as a peak shift and an accompanying

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Figure 5. Nanocapsule distension after exposure to Pluronic® surfactants (A) L62D, and (B) L44NF at: 150 mg/mL (1: 0.75) (black circles), 100 mg/mL (1:0.5) (grey diamonds), 75 mg/mL (1:0.375) (grey triangles), 50 mg/mL (1:0.25) (grey squares), and control (unfilled circles). Data represent mean ± SD, n = 3 (left). The swelling rates of the nanocapsules at each concentration of Pluronic® surfactant for (C) L62D and (D) L44NF (right).

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nanocapsule swelling response, even at high levels of the trigger, indicated that this was a robust chemically-triggered delivery system suitable for development as a trigger-responsive nanomedicine for the treatment of pulmonary disease.

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nanocapsules was thought to be enhanced by the absence of cholesterol, which has previously been shown to render lipidic barriers more resistant to Pluronic® insertion. 38 The concentration-dependent swelling responses to Pluronic® L62D and L44NF were unexpected considering that all the surfactant concentrations assessed were above the CMC of the molecules, hence any increase in concentration should theoretically generate more micelles rather than free surfactant molecules. 45 However, Pluronic® surfactants do not micellise in the same manner as conventional surfactants, their aggregation behaviour occurs over a large concentration range. 46 Furthermore, the PCS data showed that micellisation was disrupted by co-incubation of the surfactant with the nanocapsules. The influence of the surfactant aggregation process on the ability to trigger nanocapsule distension was further investigated by adjusting electrolyte concentrations. Salts such as NaCl are known to favour Pluronic® surfactant micellisation, lowering both the CMC and critical micellisation temperature. 47 -49 However, Pluronic® L62 is unusual in that its micellisation can be suppressed in the presence of NaCl. 47 PCS analysis of Pluronic® solutions revealed that L62D was most responsive to changes in electrolyte content, implying its aggregation was less stable than that of L64 and L44NF at equivalent concentrations. Electrolyte suppressed aggregation of L62D, which is more extensive for this type of Pluronic compared to the other grades, could be one of the responsible factors for its superior triggering of lipid nanocapsule shell disruption. Hyperosmolar concentrations were required to observe the electrolyte effect using the PCS assay and therefore other structural factors may also contribute to the function of L62D as an effective trigger and subsequent work using small angle light scattering could provide more insight into these interactions. Soft lipid nanocapsules engineered with a solid outer shell, could be selectively and rapidly permeabilised using the amphiphilic polymer Pluronic® L62D as a chemical trigger. The permeabilised shell of the nanocapsules was penetrable by both small hydrophilic and hydrophobic molecules and the triggering process was sufficiently selective to occur at the mucosal surface of airway epithelial cells without detrimental effects to the cells. The concentration-dependency of the

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Figure 6. (A) Change in the light scattering of Pluronic® L62D (closed squares), L64 (closed circles) and L44 NF (closed triangles) over time following dilution with water. (B) Lipid nanocapsule size (nm) following exposure to Pluronic® L62D and Pluronic® L64 (1:0.75) for 24 h as a function of NaCl content. NaCl concentrations were 0 (− NaCl), 0.9% w/v (+ NaCl) and 1.8% w/v (++NaCl). Data represent mean ± SD, n = 3.

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Appendix A. Supplementary data

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Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.nano.2014.07.012.

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Graphical Abstract Triggered-release nanocapsules for drug delivery to the lungs

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Jasminder Chana, PhD, Ben Forbes, PhD, Stuart A. Jones, PhD ⁎

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Institute of Pharmaceutical Science, King's College London, London, UK

Solid shell lipid nanocapsules where shown to distend rapidly when they incubated with Pluronic® L62. This change, triggered by the surfactant, induced a controllable capsule permeabilisation by inducing shell fluidisation. When the nanocapsules and Pluronic® L62 were applied to the air interface of human bronchial epithelial cells the nanocapsule's chemical payload was delivered effectively into the cells without deleterious effects on the barrier. The triggering mechanism was stable in complex media and the particles were shown not to induce significant inflammation in vivo in the lung thus the system was proposed as a viable delivery platform for inhaled medicines.

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Triggered-release nanocapsules for drug delivery to the lungs.

This study demonstrated the feasibility of trigger-responsive inhaled delivery of medicines using soft solid shelled nanocapsules. The delivery system...
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