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Nebulised siRNA encapsulated crosslinked chitosan nanoparticles for pulmonary delivery
ARTICLE IN PRESS
G Model IJP 13497 1–7
International Journal of Pharmaceutics xxx (2013) xxx–xxx
Contents lists available at SciVerse ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Pharmaceutical nanotechnology
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Nebulised siRNA encapsulated crosslinked chitosan nanoparticles for pulmonary delivery
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Ketan Sharma a , Satyanarayana Somavarapu a,∗ , Agnes Colombani b , Nayna Govind b , Kevin M.G. Taylor a a b
UCL, School of Pharmacy, 29-39 Brunswick Square, London WC1N 1AX, UK AstraZeneca R&D Charnwood, Bakewell Road, Loughborough LE11 5RH, UK
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Article history: Received 19 April 2013 Received in revised form 2 July 2013 Accepted 7 July 2013 Available online xxx
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Keywords: Chitosan Inhalation 18 Nanocarrier 19 Nanoparticle 20 Nebuliser 21 22 Q2 Pulmonary drug delivery siRNA 23 16 17
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Purpose: To explore the potential of crosslinked chitosan nanoparticles as carriers for delivery of siRNA using a jet nebuliser. Materials and methods: Nanoparticles encapsulating siRNA were prepared using an ionic crosslinking technique at chitosan to siRNA weight/weight ratios of 10:1, 30:1 and 50:1. Particles were characterised for their size, charge, morphology, pH stability and siRNA encapsulation efficiency. Gel electrophoresis was used to assess the association and stability of siRNA with nanoparticles, including after aerosolisation using a Pari LC Sprint jet nebuliser. The aerosolisation properties of FITC labelled chitosan nanoparticles were investigated using a two-stage impinger. Cell viability was performed with H-292 cells using a WST-1 assay. Results: Positively charged spherical nanoparticles were produced with mean diameters less than 150 nm, at all chitosan to siRNA ratios. Nanoparticles were non-aggregated at the pH of the airways and showed high siRNA encapsulation efficiency (>96%). Complete binding of siRNA to chitosan nanoparticles was observed when the w/w ratio was 50:1. Nebulisation produced fine particle fractions of 54 ± 11% and 57.3 ± 1.9% for chitosan and chitosan:siRNA (10:1 w/w) nanoparticles respectively. The stability of chitosan-encapsulated siRNA was maintained after nebulisation. Cell viability was high (>85%) at the highest chitosan concentration (83 g/ml). Conclusion: The results suggest that crosslinked chitosan nanoparticles have potential for siRNA delivery to the lungs using a jet nebuliser. © 2013 Published by Elsevier B.V.
1. Introduction In recent years, small interfering RNA (siRNA) has emerged as a therapeutic agent based on RNA interference (RNAi). The use of siRNA for sequence specific cleavage and degradation of specific mRNA is undergoing clinical trials for treatment of inheritable and infectious diseases, including pulmonary conditions (Durcan et al., 2008; Merkel and Kissel, 2012). These studies suggest that inhaled siRNA, in a properly designed formulation, might offer a fast, potent and easily administrable therapy against respiratory diseases in humans. siRNA delivery to the lungs using nebulisers offers great potential, but to date there has been little research on the aerosol properties of nebulised siRNA. During the process of nebulisation,
∗ Corresponding author at: UCL, School of Pharmacy, UK. Tel.: +44 207753 5987; fax: +44 207753 5942. E-mail addresses: [email protected], [email protected] (S. Somavarapu).
biopharmaceuticals (proteins, peptides, DNA, siRNA, etc.) may be degraded by shear stresses within the nebuliser and effects resulting from adsorption at the liquid/air interface (Niven and Brain, 1994; Khatri et al., 2001). Hence, in order to improve the stability and aerosolisation of biopharmaceuticals from nebulisers, a carrier is often needed (Birchall et al., 2000; Huth et al., 2006; Albasarah et al., 2010). A variety of carriers have been investigated for in vivo delivery of biopharmaceuticals, for instance viral vectors; adenoassociated virus, retrovirus and lentivirus, and non-viral vectors which fall into three broad categories: liposomes, polymers and peptides (Reischl and Zimmer, 2009). There has been considerable interest in formulating non-viral vectors for delivery of siRNA to the lung (Thomas et al., 2007; Merkel and Kissel, 2012), as carriers not only improve stability but also enhance cellular uptake of siRNA. Thus, while nebulisation of siRNA in its naked form resulted in considerable degradation, it was not denatured when delivered in association with the non-viral vectors, polyethylenimine (PEI) and oligofectamine (Huth et al., 2006). Chitosan has been widely investigated as a non-viral polymer for the formation of nanoparticles for delivering
0378-5173/$ – see front matter © 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.ijpharm.2013.07.024
Please cite this article in press as: Sharma, K., et al., Nebulised siRNA encapsulated crosslinked chitosan nanoparticles for pulmonary delivery. Int J Pharmaceut (2013), http://dx.doi.org/10.1016/j.ijpharm.2013.07.024
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biopharmaceuticals such as siRNA, due to its biodegradable and biocompatible properties (Howard et al., 2006; Andersen et al., 2009). In addition, its mucoadhesive and mucosa-permeation properties (Lai and Lin, 2009) make it a potentially useful carrier for pulmonary delivery. The polycationic property of chitosan allows it to bind strongly to mammalian cells and negatively charged siRNA (Lee et al., 2005) and it was shown that chitosan/DNA particles adhered to bronchiolar epithelia and facilitated gene expression in this region after intratracheal administration (Koping-Hoggard et al., 2001). Likewise, chitosan (114 kDa)/siRNA complexes showed knockdown of endogenous Enhanced Green Fluorescent Protein (EGFP) in both H1299 human lung carcinoma cells and murine peritoneal macrophages (77.9% and 89.3% reduction in EGFP fluorescence, respectively) in vitro (Howard et al., 2006). Nasal administration of these formulations showed effective in vivo RNAi in bronchiole epithelial cells of transgenic EGFP mice, with 43% reduction compared to untreated control (Howard et al., 2006; Nielsen et al., 2010). The purpose of this research work was to investigate the potential of siRNA-loaded, low molecular weight crosslinked chitosan nanoparticles for pulmonary delivery using nebulisers. The siRNA encapsulated chitosan nanoparticles formed using ionic gelation were characterised for size, charge, pH stability of carriers, siRNA encapsulation, aerosol properties and cytotoxicity.
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2. Materials and methods
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2.1. Materials
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Water soluble, low molecular weight chitosan (LMC), molecular weight 10 kDa, degree of deacetylation 97.0%, was purchased from Kittolife Co. Ltd (Korea). Fluorescein 5-isothiocyanate (FITC), tris acetate-ethylenediaminetetraacetic acid (TAE) buffer (10×), ethidium bromide, agarose, methanol, tryptan blue, 96-well plates and sodium tripolyphosphate (TPP) 85% were purchased from Sigma–Aldrich (Germany). HPLC grade water was purchased from Fischer Scientific (UK). Synthetic PTENV10-23-hmr (phosphatase and tensin homolog) with a molecular weight 7.4118 kDa and a duplex sequence (sense: 5 -UAAGUUCUAGCUGUGGUGGGUUA3 , antisense: 3 -AUUCAAGAUCGACACCACCCAAU-5 ) was obtained from Integrated DNA technologies, through AstraZeneca (UK). Gel loading dye (Blue 6×) was purchased from Biolabs (UK). PicoGreen reagent (Quant-iTTM ) was obtained from Molecular Probes (USA). Foetal bovine serum (FBS), penicillin streptomycin (Pen Strep) and ultrapure DNase/RNase-free distilled water were supplied by Invitrogen (UK). RPMI medium 1640, phosphate buffered saline (PBS) and trypsin-EDTA were purchased from Gibco (USA). H-292 cells (Part number 34) were donated by AstraZeneca (UK). WST1 reagent was obtained from Roche Diagnostics (Sweden) and donated by AstraZeneca (UK).
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2.2. Preparation of crosslinked chitosan nanoparticles
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Chitosan nanoparticles were produced using the ionic gelation method (Katas and Alpar, 2006). 2 ml TPP solution (0.5 mg/ml) was added drop wise to 10 ml chitosan solution (1 mg/ml) in the w/w ratio of 1:10 using a peristaltic pump (Gilson, France) with stirring rate of 8000 rpm at room temperature 22 ◦ C. A 10:1 chitosan to TPP ratio was based on preliminary investigation and published studies (Hou et al., 2012).
of Chae et al. (2005). 500 mg chitosan was dissolved in DMSO:water in the ratio 90:1. Separately, 20 mg of FITC (1 mg/ml) was dissolved in DMSO and added slowly to the chitosan solution. The sample was left at room temperature, in the dark for 10 h, for the chemical reaction to complete. The FITC-bound chitosan was precipitated in excess acetone (8 ml) and separated from unreacted FITC and DMSO using a Millipore filter paper. The precipitate was washed with acetone several times before freeze drying (Virtis Advantage, SP Scientific, USA) to yield FITC-labelled chitosan. The FITC content was measured at excitation and emission wavelengths of 492 nm and 518 nm respectively using a fluorescence spectrometer (LS 55, Perkin-Elmer, UK). Standard solutions of FITC, 12.5–200 ng/ml were produced. A calibration curve was generated using fluorescence spectrometry with a R2 value of 0.995. FITC-labelled chitosan was used to prepare fluorescent crosslinked nanoparticles following the protocol described in Section 2.2. 2.4. Preparation of siRNA loaded chitosan nanoparticles 166.6 l TPP solution (0.5 g/l) was mixed with siRNA (15.38 g/l) prior to nanoparticle formation. This was added dropwise to 833.3 l of chitosan solution (1 g/l) with constant stirring to form siRNA encapsulated nanoparticles. The total volume of the nanoparticles was made up to 1013.3 l with nuclease-free water. siRNA solution was added in volumes of 5.4 l, 1.8 l and 1.08 l to give chitosan to siRNA w/w ratios of 10:1, 30:1 and 50:1 respectively (these represent N:P ratios of 20:1, 60:1 and 100:1). All nanoparticle formulations were stored for 1 h prior to use in further experiments. 2.5. Size and surface charge of nanoparticles The size distribution of nanoparticles, without further dilution, was obtained as ZAve hydrodynamic diameter and polydispersity index by photon correlation spectroscopy. The surface charge of nanoparticles dispersed in deionized water was measured by laser Doppler micro-electrophoresis. These studies were performed using the ZetasizerNano ZS (Malvern Instruments, UK). 2.6. Scanning electron microscopy of nanoparticles A small amount of freeze-dried nanoparticles was placed on a scanning electron microscopy (SEM) stub and sputter-coated with gold (Emitech K550; Quorum, UK). Primary electrons were targeted over the sample and these were deflected to a secondary electron detector. This deflection is the image visualised using SEM (FEI XL30 TMP; Philips, Netherlands). 2.7. Effect of pH on nanoparticle properties The effect of pH on the size and surface charge of chitosan nanoparticles was studied using a ZetasizerNano ZS (Malvern Instruments, UK) equipped with an auto-titration unit; MPT-2 (Malvern Instruments, UK). The aqueous dispersion of chitosan nanoparticles (12 ml) was titrated with 0.1 M sodium hydroxide solution (NaOH) with constant stirring over a range of pH (5.4–8). The titrated dispersion was transferred to a measuring capillary cell by a spinning disc, and changes in the properties of the nanoparticles were measured as a function of pH. 2.8. Encapsulation efficiency of siRNA
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2.3. FITC labelled chitosan and subsequent formation of nanoparticles In order to quantify chitosan, FITC was covalently labelled to free amine groups of chitosan using a method modified from that
The encapsulation efficiency of siRNA onto or in nanoparticles was determined using the PicoGreen reagent (Quant-iTTM , Molecular Probes, USA) according to a standard protocol. 900 l of siRNA loaded nanoparticles with chitosan:siRNA ratios of 10:1,
Please cite this article in press as: Sharma, K., et al., Nebulised siRNA encapsulated crosslinked chitosan nanoparticles for pulmonary delivery. Int J Pharmaceut (2013), http://dx.doi.org/10.1016/j.ijpharm.2013.07.024
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30:1 and 50:1 (w/w) were centrifuged at 17,000 × g for 15 min at 10 ◦ C (refrigerated centrifuge Sigma 1-15PK, Osterode am Harz, Germany). For each measurement, 500 l of supernatant was mixed with 500 l of TEA (tris-hydrochloride ethylenediaminetetraacetic acid) buffer supplied with the kit and 1 ml of PicoGreen reagent (1/200 dilution with TEA buffer) to quantify free siRNA, unbound to nanoparticles. After an incubation period of 10 min, the sample was transferred to a quartz microcurvette and the amount of siRNA associated with the nanoparticles was assayed using a luminescence spectrometer (LS55, Perkin Elmer, USA) with excitation and emission wavelengths of 480 nm and 520 nm respectively. Blanks containing no siRNA were also prepared. A calibration curve ranging from 0 to 1000 ng/ml siRNA was prepared and Eq. (1) used to determine the encapsulation efficiency.
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Encapsulation efficiency =
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Total siRNA − siRNA unbound Total siRNA
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Formulation
Hydrodynamic diameter (nm ± SD)
Polydispersity index (±SD)
Zeta potential (mV ± SD)
Chitosan FITC-chitosan
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+37 ± 2 +39 ± 2
dose (RD) is defined as the sum of the total amount of chitosan determined in the nebuliser reservoir, moulded rubber mouth piece, Stages 1 and 2. Mass balance is the percentage of the recovered dose with respect to the nominal dose. Fine particle fraction (FPF) was determined as the amount of chitosan present in Stage 2 of the impinger (mass median aerodynamic diameter <6.4 m) as a percentage of the recovered dose. 2.11. Analysis of nebulised siRNA nanoparticles using gel electrophoresis
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Table 1 Particle size and surface charge of crosslinked chitosan nanoparticles and FITClabelled crosslinked chitosan nanoparticles (mean ± SD, n = 3).
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(1)
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2.9. Gel electrophoresis of nanoparticles with siRNA The stability of siRNA to various conditions encountered during the manufacture of siRNA encapsulated nanoparticles was examined by gel electrophoresis. The gel was prepared with 4% (w/v) agarose in TEA buffer. siRNA (0.2 g) was loaded onto each well, encapsulated with different ratios of chitosan as detailed in Section 2.4. The gel was run at 60 V for 1 h. siRNA movement was determined using ethidium bromide which was dissolved within the gel. Gel images were produced using a UV transilluminator image analyser (Syngene G-Box, UK). 2.10. Analysis of nebulised formulations using the two-stage impinger Formulations were aerosolised using a jet nebuliser into a twostage impinger (TSI; Copley Scientific Ltd., UK), using the procedure described in the European Pharmacopoeia (2008). Nanoparticles suspended in 5 ml aqueous medium (water) were placed in the nebuliser reservoir (Pari LC Sprint nebuliser, Pari Gmbh, Germany). The formulations contained chitosan at a concentration of 833.3 g/ml. The nebuliser reservoir was attached to the TSI via a moulded rubber mouthpiece and aerosols were generated using a Turbo Boy N compressor (Pari Gmbh, Germany). The aerosolised nanoparticles passed into the TSI which contained deionised water in the upper stage (Stage 1, 7 ml) and lower stage (Stage 2, 30 ml). The flow rate was set at 60 L/min using a digital flow metre (DFM 2000, Copley Scientific Ltd., UK) achieved by a vacuum pump (HCP 5, Copley Scientific Ltd., UK). The TSI was covered with aluminium foil to prevent the degradation of FITC by light. Towards the end of nebulisation, the nebuliser was tapped to maximise fluid output. The aerosolised formulation was collected in Stages 1 and 2 of TSI which was dismantled and the stages, the moulded rubber mouthpiece and the nebuliser reservoir (containing residual fluid) were washed separately with deionised water (in the dark) and made up to the desired volume in a volumetric flask. These samples were then analysed for their fluorescence (max excitation = 492 nm, max emission = 518 nm) using a fluorescence spectrometer (LS 55, Perkin-Elmer, UK). Based on the fluorescence in each stage, the concentration of FITC was calculated by reference to a standard curve prepared from serial dilutions of a stock solution of FITC in water. FITC concentration was converted to chitosan concentration using the previously calculated FITC percentage content in labelled chitosan. The amount of chitosan initially added to the nebuliser reservoir was considered the nominal dose or original dose. The recovered
siRNA (81.9 g/ml) encapsulated within chitosan nanoparticles (FITC-chitosan 833.3 g/ml) with a chitosan to siRNA ratio of 10:1 w/w were nebulised (5 ml) into the TSI using the methodology described in Section 2.10. The liquid was collected from Stage 2 and centrifuged at 17,000 × g for 30 min (Sigma 1-15PK, Osterode am Harz, Germany). The pellet was re-suspended in 4 ml DNAse and RNAse free water and assessed for siRNA stability on 4% (w/v) agarose gel dispersed within TAE buffer (Section 2.9). The sample was run at 60 V for 45 min and visualised using a UV transilluminator image analyser (Syngene G-Box, UK). 2.12. Influence of nanoparticles on cellular viability H-292 cells (lung muco-epithelial cells) were incubated (Heracell, USA) at 37 ◦ C and 5% CO2 overnight in 100 l RPMI media which consisted of 10% FBS, 1% Pen Strep and 1% glutamine at a seed density of 1 × 104 cells per well. The media was removed and replenished with fresh media (100 l) containing nanoparticle formulation/siRNA (chitosan to siRNA w/w ratio of 30:1) which were prepared aseptically and placed for 24 h in an incubator (37 ◦ C and 5% CO2 ). The concentration of chitosan ranged from 2.6 to 83.3 g/ml. After 24 h, the incubation media was aspirated off the cells. 10% WST-1 reagent along with media was added to each well and incubated for 15 min. WST-1 is reduced to form soluble coloured formazan products by dehydrogenase present in intact mitochondria. Absorbance of the formazan product was measured at a wavelength of 540 nm using a microplate reader (SPECTRAmax M5; Molecular devices, USA). 2.13. Statistical analysis The data are presented as a mean ± standard deviation. The samples were compared statistically using a non-parametric Kruskal–Wallis test followed by a Nemenyis post hoc test. The results were considered significantly different based upon 95% probability values (p < 0.05).
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3. Results
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3.1. Size and surface charge of chitosan nanoparticles
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Crosslinked chitosan nanoparticles had a mean hydrodynamic diameter of 123 nm (Table 1). The size distribution was unimodal with a polydispersity index (PDI) of 0.19 and the surface charge was positive. The FITC percentage content was found to be 3.51%
Please cite this article in press as: Sharma, K., et al., Nebulised siRNA encapsulated crosslinked chitosan nanoparticles for pulmonary delivery. Int J Pharmaceut (2013), http://dx.doi.org/10.1016/j.ijpharm.2013.07.024
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Table 2 Particle size and surface charge of chitosan:siRNA nanoparticles, in ratio 10:1, 30:1 and 50:1 w/w (mean ± SD, n = 3).
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Hydrodynamic diameter (nm ± SD)
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(w/w) with a labelling efficiency of 91%. The size and surface charge properties of the FITC-labelled chitosan formulation was not significantly different (p > 0.05) from the corresponding unlabelled formulation (Table 1), indicating that FITC labelling did not alter these important parameters, probably due to the low degree of conjugation and low molecular weight of FITC.
Inclusion of siRNA within chitosan nanoparticle formulations at various ratios did not produce a significant difference (p > 0.05) in mean nanoparticle particle size (Table 2), with the mean particle size for all formulations being less than 140 nm. With a higher amount of siRNA, i.e. chitosan to siRNA in ratio of 10:1 (w/w), the polydispersity index was 0.22 whereas with less siRNA, the polydispersity index was smaller. The surface charge of siRNA loaded nanoparticles ranged from +35 to +41 mV (Table 2). These values were not significantly different (p > 0.05) when compared to unloaded labelled and unlabelled nanoparticles (Table 1). Comparing siRNA-loaded formulations in different ratios, in general as the siRNA loading decreased, a higher surface charge was obtained. 3.3. Scanning electron microscopy of nanoparticles
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3.4. Effect of pH on nanoparticle properties
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In order to explore the potential of crosslinked chitosan nanoparticles for pulmonary delivery, it is important to consider
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their behaviour at lung pH, i.e. pH 6.5 (Mohri et al., 2010). As there is no change in size and surface charge of siRNA loaded chitosan nanoparticles for these studies unloaded chitosan nanoparticles are used for pH stability studies. The change in surface charge of nanoparticles over the pH range 5.4–8.0 is shown in Fig. 2. In acidic pH, 5.4, the nanoparticles had a maximum surface charge of +46 mV. The surface charge reduced as the pH increased. The nanoparticles were still positively charged even at a pH as high as 7.9. The change in hydrodynamic diameter of chitosan nanoparticles at different pH values (5.4–8.0) is shown in Fig. 3. Over the pH range 5.4–6.8, nanoparticle size remained constant, with mean hydrodynamic diameters less than 200 nm. The size increased to greater than 1 m when the pH of the medium was raised above 7.4, larger than can be accurately measured by dynamic light scattering. With increased pH, particle aggregation visibly occurred, leading to a turbid dispersion. 3.5. Encapsulation efficiency of nanoparticles
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An encapsulation efficiency of >96% was achieved for chitosan:siRNA nanoparticles in all ratios (Fig. 4). When comparing
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3.2. Size and surface charge of siRNA encapsulated nanoparticles
Following freeze-drying, SEM of fluorescent labelled particles showed spherical nanosized structures with almost uniform size distribution (Fig. 1). Similar observations were made for unlabelled nanoparticles. These fluorescent chitosan nanoparticles were used in further studies.
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Zeta potenal (mV)
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Fig. 3. Influence of pH on the hydrodynamic diameter of crosslinked chitosan nanoparticles (mean ± SD, n = 4).
Please cite this article in press as: Sharma, K., et al., Nebulised siRNA encapsulated crosslinked chitosan nanoparticles for pulmonary delivery. Int J Pharmaceut (2013), http://dx.doi.org/10.1016/j.ijpharm.2013.07.024
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Fig. 4. Encapsulation efficiency of siRNA in chitosan:siRNA nanoparticles at ratio of 10:1, 30:1 and 50:1 w/w (mean ± SD, n = 3).
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siRNA-loaded nanoparticle formulations in w/w ratios of 10:1, 30:1 and 50:1, no significant difference (p > 0.05) in encapsulation efficiency was observed.
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3.6. siRNA binding affinity with chitosan nanoparticles
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Following gel electrophoresis, negatively charged free siRNA (Lane 1) migrated towards the positive electrode (Fig. 5). For chitosan:siRNA (10:1) (Lane 2), siRNA is present in a high amount with respect to the formulation and cannot to be retained completely encapsulated within the nanoparticles in the well, and consequently the siRNA moved to the same level as the control siRNA (Lane 1). At a ratio of 30:1 (Lane 3), siRNA remained almost completely within the well, with a slight ‘smear’ away from the well. At the highest ratio of 50:1 (Lane 4), complete siRNA binding was observed, with the nanoparticles retaining siRNA within the well. This suggests that as the amount of chitosan increased in the nanoparticle, binding affinity improves. Poor retention of siRNA within crosslinked chitosan (20 kDa):siRNA nanoparticles at a weight ratio of 80:1 has been described previously (Rojanarata et al., 2008).
Fig. 6. Chitosan deposited on each stage of the TSI and remaining in the nebuliser following nebulisation of FITC-labelled chitosan nanoparticles (with and without siRNA) (mean ± SD, n = 3).
3.7. Nebulisation of unloaded and siRNA-loaded chitosan nanoparticles into the TSI In vitro analysis of the aerosol performance of nanoparticles delivered from a Pari LC Sprint jet nebuliser showed that a higher proportion of chitosan was deposited on Stage 2, than Stage 1 of the TSI, indicating suitable aerosolisation properties for lung delivery (Fig. 6). Nebulised chitosan nanoparticles had a FPF of 54 ± 11% in the TSI. siRNA-loaded chitosan nanoparticles (chitosan: siRNA ratio of 10:1 w/w) had a comparable FPF of 57.3 ± 1.9% (p > 0.05), indicating that the presence of siRNA within the system (even at a relatively high concentration) did not affect the aerosolisation behaviour of nanoparticles. Chitosan recovered from the nebuliser, and all parts of TSI, achieved a mass balance of 91 ± 7% for chitosan nanoparticle formulation and 92.9 ± 1.8% for chitosan:siRNA (10:1 w/w) nanoparticle formulation, within the European Pharmacopoeia (2008) limits of 75–125%. 3.8. Gel electrophoresis following nebulisation of siRNA-loaded nanoparticles Following aerosolisation and collection of deposited material from Stage 2 of the TSI, naked siRNA could not be detected in Lane 1 following gel electrophoresis, indicating either siRNA degradation during aerosolisation or an inability to form aerosols by nebulisation (Fig. 7). The slight band in Lane 1 was the side numbering on the gel plate present by default. Pre-nebulised, chitosan:siRNA (Lane 2) nanoparticles in the w/w ratio of 10:1 showed a bright clear band mostly moving away from the well, with slight retention within the well. This was due to the relatively high ratio of negatively charged siRNA to positively charged chitosan. These results are the same as shown in Fig. 5. Following nebulisation of chitosan:siRNA nanoparticles (Lane 3), the siRNA behaved in the gel in a similar manner to the same formulation prior to nebulisation (Lane 2). The band intensity of post-nebulised siRNA observed in gel electrophoresis was less than chitosan:siRNA nanoparticles before nebulisation, due to dilution of samples within the impinger. These findings signify stability and successful aerosolisation of siRNA when associated with the chitosan oligosaccharide nanoparticles. 3.9. In vitro assay of siRNA:chitosan nanoparticle cell viability
Fig. 5. Gel electrophoresis of siRNA and siRNA encapsulated nanoparticles. Lane 1: Naked siRNA (control); Lanes 2–4: chitosan:siRNA nanoparticles at w/w ratio of 10:1, 30:1 and 50:1 respectively.
siRNA-loaded nanoparticles at a chitosan nanoparticle concentration of 2.6 g/ml did not show cellular toxicity (H-292 cells) over a period of 24 h (Fig. 8), comparable with non-treated cells (no formulation present). At the highest chitosan nanoparticle
Please cite this article in press as: Sharma, K., et al., Nebulised siRNA encapsulated crosslinked chitosan nanoparticles for pulmonary delivery. Int J Pharmaceut (2013), http://dx.doi.org/10.1016/j.ijpharm.2013.07.024
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Fig. 7. Gel electrophoresis of siRNA before and after nebulisation, with and without chitosan, collected from Stage 2 of the TSI; Lane 1: Naked siRNA (control) after nebulisation, Lane 2: chitosan:siRNA (10:1 w/w) before nebulisation, Lane 3: chitosan:siRNA (10:1 w/w) after nebulisation.
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Amount of chitosan in each well (μg/ml) Fig. 8. H-292 cell viability following exposure to nanoparticles with chitosan (encapsulated siRNA in ratio of 30:1 w/w) ranging from 2.6 to 83.3 g/ml over a 24 h incubation period (mean ± SD, n = 3).
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concentration (83 g/ml) the cell viability reduced to 85%. These observations collectively demonstrate that chitosan nanoparticles loaded with siRNA, at the chitosan concentrations and siRNA ratios studied here do not affect cell viability and may be used for in vivo applications.
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4. Discussion
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The ionic interaction between the positively charged amine groups of chitosan and the negatively charged phosphate groups of TPP generated crosslinked nanoparticles. The cationic nature of these nanoparticles makes them useful for production of complexes with negatively charged siRNA for biopharmaceutical applications and for promoting high transfection efficiency within negatively charged cells, as a result of electrostatic interactions (Howard et al., 2006; Andersen et al., 2009). The surface charge of chitosan nanoparticles is dependent on pH. As the pH of the medium in which these nanoparticles were dispersed was increased, a
greater proportion of amine groups were deprotonated resulting in a decrease in the measured positive surface charge, reducing steric hindrance between particles, resulting in particle aggregation. It was observed that the physical stability of the crosslinked chitosan nanoparticles was pH dependent, but considerable aggregation did not occur at the physiological pH of the lung; i.e. 6.5. The changes in surface charge and hydrodynamic diameter correlated well over the pH range 5.4–8, in agreement with a study of chitosanenoxaparin complexes which were stable in the pH range 3–6.5, but aggregated at higher pH (Sun et al., 2008). One of the main issues with non-viral carriers is maintaining the original particle size in biological media. These studies show that the chitosan nanoparticles were physically stable at the pH likely to be encountered at their site of deposition following nebulisation. The encapsulation efficiency of a biopharmaceutical into chitosan nanoparticles is dependent on several parameters, including the molecular weight of chitosan, the nature of the biopharmaceutical, stirring speed, formulation composition, concentration and volume ratio of TPP and type of chitosan used. The methodology employed here resulted in high encapsulation efficiency (>96%) of siRNA into crosslinked chitosan nanoparticles. In a previous study, chitosan (50 k–150 kDa) nanoparticles prepared using TPP as an ionic crosslinker, encapsulating siRNA at an N:P ratio of 30:1, showed a similar encapsulation efficiency of >95% (Dehousse et al., 2010). Gel electrophoresis was performed to assess the association (encapsulation) of siRNA within nanoparticles. A clear bright uniform band was visualised. Upon siRNA interaction with chitosan the positively charged nanoparticles retarded the movement of negatively charged siRNA towards the positive electrode. The bright band of siRNA is an indication that the siRNA nanoparticle preparation was successful, maintaining the integrity of siRNA during the stage of encapsulation. Preliminary experiments were performed to investigate both high molecular chitosan (HMC; glutamate derivative) and low molecular weight chitosan (LMC) nanoparticles as potential carriers for pulmonary delivery. It was observed that upon their nebulisation the FPF for both was not significantly different (results not shown). Since LMC was a non-modified chitosan unlike HMC which was a glutamate derivative, LMC was selected for the further experiments described here. Following nebulisation from a jet nebuliser, chitosan:siRNA (10:1 w/w) nanoparticles showed a high nanoparticle deposition in Stage 2 of the TSI (57.3 ± 1.9% FPF) and less deposition in Stage 1, with a proportion of nanoparticles remaining in the residual fluid that remains in the nebuliser following nebulisation. A 10:1 ratio was considered appropriate in this experiment, since at this ratio the siRNA was partially bound and unbound to the carrier and so might be predicted to release siRNA during nebulisation, possibly resulting in denaturation, and following delivery, which is therapeutically desirable. Aerosols reaching Stage 2 of the TSI have mass median aerodynamic diameter (MMAD) <6.4 m and are predicted to reach the peripheral region of the lung. This suggests that the formulation is suitable as a carrier for delivery to the lower respiratory tract. In order to investigate the integrity of siRNA following nebulisation, the formulation deposited in Stage 2 was studied using gel electrophoresis. Following nebulisation, naked siRNA did not show any band, whilst nebulised siRNA encapsulated within nanoparticles showed a siRNA band on the gel. The stability of biopharmaceuticals may be compromised during jet nebulisation as a result of shearing forces in the nebuliser and surface effects at the liquid droplet/air interface (Niven and Brain, 1994). Association with the nanoparticles has ensured the integrity of siRNA is maintained during nebulisation. Previously, complexation of siRNA with positively charged PEI was found to be necessary to maintain stability during nebulisation (Huth et al., 2006). These findings, thus, confirm successful siRNA aerosolisation using a nebuliser and delivery to Stage
Please cite this article in press as: Sharma, K., et al., Nebulised siRNA encapsulated crosslinked chitosan nanoparticles for pulmonary delivery. Int J Pharmaceut (2013), http://dx.doi.org/10.1016/j.ijpharm.2013.07.024
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2 of the TSI using a new class of chitosan nanoparticle formulation, indicating its potential for in vivo studies. Nanoparticles delivered to the lungs come into contact with epithelial cells lining the respiratory tract. One of the main concerns with non-viral vectors is toxicity and hence it is essential to determine the cytotoxicity of the siRNA-nanoparticle formulation towards the target site. Nanoparticles prepared at chitosan:siRNA w/w ratios of 30:1 were selected for cytotoxicity studies, because at this ratio particles showed almost complete binding to the siRNA, had an appropriate size and positive surface charge. These nanoparticles had no overt toxicity to H-292 cells following exposure at chitosan concentration of up to 83.3 g/ml (24 h). Previously, chitosan has been shown to exhibit molecular weight-dependent cytotoxic effects, with water soluble chitosan (<10 kDa) at a low concentration (<1 mg/ml) showing negligible cytotoxic effect on the Caco-2 cells (incubated over 2 h) (Chae et al., 2005). The use of chitosan in the delivery of nucleic acids is well established (Lee et al., 2001; Lai and Lin, 2009). The results obtained here are encouraging with respect to the development of crosslinked chitosan nanoparticles for pulmonary administration of siRNA. The formulation possesses suitable aerodynamic characteristics, physical stability, the ability to encapsulate siRNA and in vitro biocompatibility, as demonstrated previously (Mwale et al., 2005). 5. Conclusion
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Nebulisation of biopharmaceuticals in solution may result in their degradation. Their stability can be improved by association or complexation with carrier particles. In this study, siRNA was successfully associated with non-toxic, crosslinked chitosan nanoparticles of appropriate size and charge, which protected the siRNA against the physical stresses encountered during nebulisation The combined features of nanoparticle stability at the pH of lung airways, high encapsulation efficiency, low cytotoxicity and efficient aerosolisation suggest these biodegradable chitosan nanoparticles would be a suitable siRNA carrier for future in vivo studies.
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Financial support from AstraZeneca is gratefully acknowledged. We also thank David McCarthy, Hamid Ali Merchant and Jonathan David Curry of UCL School of Pharmacy for technical advice.
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Please cite this article in press as: Sharma, K., et al., Nebulised siRNA encapsulated crosslinked chitosan nanoparticles for pulmonary delivery. Int J Pharmaceut (2013), http://dx.doi.org/10.1016/j.ijpharm.2013.07.024
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Contents lists available at SciVerse ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Pharmaceutical nanotechnology
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Nebulised siRNA encapsulated crosslinked chitosan nanoparticles for pulmonary delivery
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Ketan Sharma a , Satyanarayana Somavarapu a,∗ , Agnes Colombani b , Nayna Govind b , Kevin M.G. Taylor a a b
UCL, School of Pharmacy, 29-39 Brunswick Square, London WC1N 1AX, UK AstraZeneca R&D Charnwood, Bakewell Road, Loughborough LE11 5RH, UK
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Article history: Received 19 April 2013 Received in revised form 2 July 2013 Accepted 7 July 2013 Available online xxx
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Keywords: Chitosan Inhalation 18 Nanocarrier 19 Nanoparticle 20 Nebuliser 21 22 Q2 Pulmonary drug delivery siRNA 23 16 17
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Purpose: To explore the potential of crosslinked chitosan nanoparticles as carriers for delivery of siRNA using a jet nebuliser. Materials and methods: Nanoparticles encapsulating siRNA were prepared using an ionic crosslinking technique at chitosan to siRNA weight/weight ratios of 10:1, 30:1 and 50:1. Particles were characterised for their size, charge, morphology, pH stability and siRNA encapsulation efficiency. Gel electrophoresis was used to assess the association and stability of siRNA with nanoparticles, including after aerosolisation using a Pari LC Sprint jet nebuliser. The aerosolisation properties of FITC labelled chitosan nanoparticles were investigated using a two-stage impinger. Cell viability was performed with H-292 cells using a WST-1 assay. Results: Positively charged spherical nanoparticles were produced with mean diameters less than 150 nm, at all chitosan to siRNA ratios. Nanoparticles were non-aggregated at the pH of the airways and showed high siRNA encapsulation efficiency (>96%). Complete binding of siRNA to chitosan nanoparticles was observed when the w/w ratio was 50:1. Nebulisation produced fine particle fractions of 54 ± 11% and 57.3 ± 1.9% for chitosan and chitosan:siRNA (10:1 w/w) nanoparticles respectively. The stability of chitosan-encapsulated siRNA was maintained after nebulisation. Cell viability was high (>85%) at the highest chitosan concentration (83 g/ml). Conclusion: The results suggest that crosslinked chitosan nanoparticles have potential for siRNA delivery to the lungs using a jet nebuliser. © 2013 Published by Elsevier B.V.
1. Introduction In recent years, small interfering RNA (siRNA) has emerged as a therapeutic agent based on RNA interference (RNAi). The use of siRNA for sequence specific cleavage and degradation of specific mRNA is undergoing clinical trials for treatment of inheritable and infectious diseases, including pulmonary conditions (Durcan et al., 2008; Merkel and Kissel, 2012). These studies suggest that inhaled siRNA, in a properly designed formulation, might offer a fast, potent and easily administrable therapy against respiratory diseases in humans. siRNA delivery to the lungs using nebulisers offers great potential, but to date there has been little research on the aerosol properties of nebulised siRNA. During the process of nebulisation,
∗ Corresponding author at: UCL, School of Pharmacy, UK. Tel.: +44 207753 5987; fax: +44 207753 5942. E-mail addresses: [email protected], [email protected] (S. Somavarapu).
biopharmaceuticals (proteins, peptides, DNA, siRNA, etc.) may be degraded by shear stresses within the nebuliser and effects resulting from adsorption at the liquid/air interface (Niven and Brain, 1994; Khatri et al., 2001). Hence, in order to improve the stability and aerosolisation of biopharmaceuticals from nebulisers, a carrier is often needed (Birchall et al., 2000; Huth et al., 2006; Albasarah et al., 2010). A variety of carriers have been investigated for in vivo delivery of biopharmaceuticals, for instance viral vectors; adenoassociated virus, retrovirus and lentivirus, and non-viral vectors which fall into three broad categories: liposomes, polymers and peptides (Reischl and Zimmer, 2009). There has been considerable interest in formulating non-viral vectors for delivery of siRNA to the lung (Thomas et al., 2007; Merkel and Kissel, 2012), as carriers not only improve stability but also enhance cellular uptake of siRNA. Thus, while nebulisation of siRNA in its naked form resulted in considerable degradation, it was not denatured when delivered in association with the non-viral vectors, polyethylenimine (PEI) and oligofectamine (Huth et al., 2006). Chitosan has been widely investigated as a non-viral polymer for the formation of nanoparticles for delivering
0378-5173/$ – see front matter © 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.ijpharm.2013.07.024
Please cite this article in press as: Sharma, K., et al., Nebulised siRNA encapsulated crosslinked chitosan nanoparticles for pulmonary delivery. Int J Pharmaceut (2013), http://dx.doi.org/10.1016/j.ijpharm.2013.07.024
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biopharmaceuticals such as siRNA, due to its biodegradable and biocompatible properties (Howard et al., 2006; Andersen et al., 2009). In addition, its mucoadhesive and mucosa-permeation properties (Lai and Lin, 2009) make it a potentially useful carrier for pulmonary delivery. The polycationic property of chitosan allows it to bind strongly to mammalian cells and negatively charged siRNA (Lee et al., 2005) and it was shown that chitosan/DNA particles adhered to bronchiolar epithelia and facilitated gene expression in this region after intratracheal administration (Koping-Hoggard et al., 2001). Likewise, chitosan (114 kDa)/siRNA complexes showed knockdown of endogenous Enhanced Green Fluorescent Protein (EGFP) in both H1299 human lung carcinoma cells and murine peritoneal macrophages (77.9% and 89.3% reduction in EGFP fluorescence, respectively) in vitro (Howard et al., 2006). Nasal administration of these formulations showed effective in vivo RNAi in bronchiole epithelial cells of transgenic EGFP mice, with 43% reduction compared to untreated control (Howard et al., 2006; Nielsen et al., 2010). The purpose of this research work was to investigate the potential of siRNA-loaded, low molecular weight crosslinked chitosan nanoparticles for pulmonary delivery using nebulisers. The siRNA encapsulated chitosan nanoparticles formed using ionic gelation were characterised for size, charge, pH stability of carriers, siRNA encapsulation, aerosol properties and cytotoxicity.
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2. Materials and methods
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2.1. Materials
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Water soluble, low molecular weight chitosan (LMC), molecular weight 10 kDa, degree of deacetylation 97.0%, was purchased from Kittolife Co. Ltd (Korea). Fluorescein 5-isothiocyanate (FITC), tris acetate-ethylenediaminetetraacetic acid (TAE) buffer (10×), ethidium bromide, agarose, methanol, tryptan blue, 96-well plates and sodium tripolyphosphate (TPP) 85% were purchased from Sigma–Aldrich (Germany). HPLC grade water was purchased from Fischer Scientific (UK). Synthetic PTENV10-23-hmr (phosphatase and tensin homolog) with a molecular weight 7.4118 kDa and a duplex sequence (sense: 5 -UAAGUUCUAGCUGUGGUGGGUUA3 , antisense: 3 -AUUCAAGAUCGACACCACCCAAU-5 ) was obtained from Integrated DNA technologies, through AstraZeneca (UK). Gel loading dye (Blue 6×) was purchased from Biolabs (UK). PicoGreen reagent (Quant-iTTM ) was obtained from Molecular Probes (USA). Foetal bovine serum (FBS), penicillin streptomycin (Pen Strep) and ultrapure DNase/RNase-free distilled water were supplied by Invitrogen (UK). RPMI medium 1640, phosphate buffered saline (PBS) and trypsin-EDTA were purchased from Gibco (USA). H-292 cells (Part number 34) were donated by AstraZeneca (UK). WST1 reagent was obtained from Roche Diagnostics (Sweden) and donated by AstraZeneca (UK).
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2.2. Preparation of crosslinked chitosan nanoparticles
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Chitosan nanoparticles were produced using the ionic gelation method (Katas and Alpar, 2006). 2 ml TPP solution (0.5 mg/ml) was added drop wise to 10 ml chitosan solution (1 mg/ml) in the w/w ratio of 1:10 using a peristaltic pump (Gilson, France) with stirring rate of 8000 rpm at room temperature 22 ◦ C. A 10:1 chitosan to TPP ratio was based on preliminary investigation and published studies (Hou et al., 2012).
of Chae et al. (2005). 500 mg chitosan was dissolved in DMSO:water in the ratio 90:1. Separately, 20 mg of FITC (1 mg/ml) was dissolved in DMSO and added slowly to the chitosan solution. The sample was left at room temperature, in the dark for 10 h, for the chemical reaction to complete. The FITC-bound chitosan was precipitated in excess acetone (8 ml) and separated from unreacted FITC and DMSO using a Millipore filter paper. The precipitate was washed with acetone several times before freeze drying (Virtis Advantage, SP Scientific, USA) to yield FITC-labelled chitosan. The FITC content was measured at excitation and emission wavelengths of 492 nm and 518 nm respectively using a fluorescence spectrometer (LS 55, Perkin-Elmer, UK). Standard solutions of FITC, 12.5–200 ng/ml were produced. A calibration curve was generated using fluorescence spectrometry with a R2 value of 0.995. FITC-labelled chitosan was used to prepare fluorescent crosslinked nanoparticles following the protocol described in Section 2.2. 2.4. Preparation of siRNA loaded chitosan nanoparticles 166.6 l TPP solution (0.5 g/l) was mixed with siRNA (15.38 g/l) prior to nanoparticle formation. This was added dropwise to 833.3 l of chitosan solution (1 g/l) with constant stirring to form siRNA encapsulated nanoparticles. The total volume of the nanoparticles was made up to 1013.3 l with nuclease-free water. siRNA solution was added in volumes of 5.4 l, 1.8 l and 1.08 l to give chitosan to siRNA w/w ratios of 10:1, 30:1 and 50:1 respectively (these represent N:P ratios of 20:1, 60:1 and 100:1). All nanoparticle formulations were stored for 1 h prior to use in further experiments. 2.5. Size and surface charge of nanoparticles The size distribution of nanoparticles, without further dilution, was obtained as ZAve hydrodynamic diameter and polydispersity index by photon correlation spectroscopy. The surface charge of nanoparticles dispersed in deionized water was measured by laser Doppler micro-electrophoresis. These studies were performed using the ZetasizerNano ZS (Malvern Instruments, UK). 2.6. Scanning electron microscopy of nanoparticles A small amount of freeze-dried nanoparticles was placed on a scanning electron microscopy (SEM) stub and sputter-coated with gold (Emitech K550; Quorum, UK). Primary electrons were targeted over the sample and these were deflected to a secondary electron detector. This deflection is the image visualised using SEM (FEI XL30 TMP; Philips, Netherlands). 2.7. Effect of pH on nanoparticle properties The effect of pH on the size and surface charge of chitosan nanoparticles was studied using a ZetasizerNano ZS (Malvern Instruments, UK) equipped with an auto-titration unit; MPT-2 (Malvern Instruments, UK). The aqueous dispersion of chitosan nanoparticles (12 ml) was titrated with 0.1 M sodium hydroxide solution (NaOH) with constant stirring over a range of pH (5.4–8). The titrated dispersion was transferred to a measuring capillary cell by a spinning disc, and changes in the properties of the nanoparticles were measured as a function of pH. 2.8. Encapsulation efficiency of siRNA
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2.3. FITC labelled chitosan and subsequent formation of nanoparticles In order to quantify chitosan, FITC was covalently labelled to free amine groups of chitosan using a method modified from that
The encapsulation efficiency of siRNA onto or in nanoparticles was determined using the PicoGreen reagent (Quant-iTTM , Molecular Probes, USA) according to a standard protocol. 900 l of siRNA loaded nanoparticles with chitosan:siRNA ratios of 10:1,
Please cite this article in press as: Sharma, K., et al., Nebulised siRNA encapsulated crosslinked chitosan nanoparticles for pulmonary delivery. Int J Pharmaceut (2013), http://dx.doi.org/10.1016/j.ijpharm.2013.07.024
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30:1 and 50:1 (w/w) were centrifuged at 17,000 × g for 15 min at 10 ◦ C (refrigerated centrifuge Sigma 1-15PK, Osterode am Harz, Germany). For each measurement, 500 l of supernatant was mixed with 500 l of TEA (tris-hydrochloride ethylenediaminetetraacetic acid) buffer supplied with the kit and 1 ml of PicoGreen reagent (1/200 dilution with TEA buffer) to quantify free siRNA, unbound to nanoparticles. After an incubation period of 10 min, the sample was transferred to a quartz microcurvette and the amount of siRNA associated with the nanoparticles was assayed using a luminescence spectrometer (LS55, Perkin Elmer, USA) with excitation and emission wavelengths of 480 nm and 520 nm respectively. Blanks containing no siRNA were also prepared. A calibration curve ranging from 0 to 1000 ng/ml siRNA was prepared and Eq. (1) used to determine the encapsulation efficiency.
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Encapsulation efficiency =
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Total siRNA − siRNA unbound Total siRNA
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Formulation
Hydrodynamic diameter (nm ± SD)
Polydispersity index (±SD)
Zeta potential (mV ± SD)
Chitosan FITC-chitosan
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dose (RD) is defined as the sum of the total amount of chitosan determined in the nebuliser reservoir, moulded rubber mouth piece, Stages 1 and 2. Mass balance is the percentage of the recovered dose with respect to the nominal dose. Fine particle fraction (FPF) was determined as the amount of chitosan present in Stage 2 of the impinger (mass median aerodynamic diameter <6.4 m) as a percentage of the recovered dose. 2.11. Analysis of nebulised siRNA nanoparticles using gel electrophoresis
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Table 1 Particle size and surface charge of crosslinked chitosan nanoparticles and FITClabelled crosslinked chitosan nanoparticles (mean ± SD, n = 3).
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(1)
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2.9. Gel electrophoresis of nanoparticles with siRNA The stability of siRNA to various conditions encountered during the manufacture of siRNA encapsulated nanoparticles was examined by gel electrophoresis. The gel was prepared with 4% (w/v) agarose in TEA buffer. siRNA (0.2 g) was loaded onto each well, encapsulated with different ratios of chitosan as detailed in Section 2.4. The gel was run at 60 V for 1 h. siRNA movement was determined using ethidium bromide which was dissolved within the gel. Gel images were produced using a UV transilluminator image analyser (Syngene G-Box, UK). 2.10. Analysis of nebulised formulations using the two-stage impinger Formulations were aerosolised using a jet nebuliser into a twostage impinger (TSI; Copley Scientific Ltd., UK), using the procedure described in the European Pharmacopoeia (2008). Nanoparticles suspended in 5 ml aqueous medium (water) were placed in the nebuliser reservoir (Pari LC Sprint nebuliser, Pari Gmbh, Germany). The formulations contained chitosan at a concentration of 833.3 g/ml. The nebuliser reservoir was attached to the TSI via a moulded rubber mouthpiece and aerosols were generated using a Turbo Boy N compressor (Pari Gmbh, Germany). The aerosolised nanoparticles passed into the TSI which contained deionised water in the upper stage (Stage 1, 7 ml) and lower stage (Stage 2, 30 ml). The flow rate was set at 60 L/min using a digital flow metre (DFM 2000, Copley Scientific Ltd., UK) achieved by a vacuum pump (HCP 5, Copley Scientific Ltd., UK). The TSI was covered with aluminium foil to prevent the degradation of FITC by light. Towards the end of nebulisation, the nebuliser was tapped to maximise fluid output. The aerosolised formulation was collected in Stages 1 and 2 of TSI which was dismantled and the stages, the moulded rubber mouthpiece and the nebuliser reservoir (containing residual fluid) were washed separately with deionised water (in the dark) and made up to the desired volume in a volumetric flask. These samples were then analysed for their fluorescence (max excitation = 492 nm, max emission = 518 nm) using a fluorescence spectrometer (LS 55, Perkin-Elmer, UK). Based on the fluorescence in each stage, the concentration of FITC was calculated by reference to a standard curve prepared from serial dilutions of a stock solution of FITC in water. FITC concentration was converted to chitosan concentration using the previously calculated FITC percentage content in labelled chitosan. The amount of chitosan initially added to the nebuliser reservoir was considered the nominal dose or original dose. The recovered
siRNA (81.9 g/ml) encapsulated within chitosan nanoparticles (FITC-chitosan 833.3 g/ml) with a chitosan to siRNA ratio of 10:1 w/w were nebulised (5 ml) into the TSI using the methodology described in Section 2.10. The liquid was collected from Stage 2 and centrifuged at 17,000 × g for 30 min (Sigma 1-15PK, Osterode am Harz, Germany). The pellet was re-suspended in 4 ml DNAse and RNAse free water and assessed for siRNA stability on 4% (w/v) agarose gel dispersed within TAE buffer (Section 2.9). The sample was run at 60 V for 45 min and visualised using a UV transilluminator image analyser (Syngene G-Box, UK). 2.12. Influence of nanoparticles on cellular viability H-292 cells (lung muco-epithelial cells) were incubated (Heracell, USA) at 37 ◦ C and 5% CO2 overnight in 100 l RPMI media which consisted of 10% FBS, 1% Pen Strep and 1% glutamine at a seed density of 1 × 104 cells per well. The media was removed and replenished with fresh media (100 l) containing nanoparticle formulation/siRNA (chitosan to siRNA w/w ratio of 30:1) which were prepared aseptically and placed for 24 h in an incubator (37 ◦ C and 5% CO2 ). The concentration of chitosan ranged from 2.6 to 83.3 g/ml. After 24 h, the incubation media was aspirated off the cells. 10% WST-1 reagent along with media was added to each well and incubated for 15 min. WST-1 is reduced to form soluble coloured formazan products by dehydrogenase present in intact mitochondria. Absorbance of the formazan product was measured at a wavelength of 540 nm using a microplate reader (SPECTRAmax M5; Molecular devices, USA). 2.13. Statistical analysis The data are presented as a mean ± standard deviation. The samples were compared statistically using a non-parametric Kruskal–Wallis test followed by a Nemenyis post hoc test. The results were considered significantly different based upon 95% probability values (p < 0.05).
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Crosslinked chitosan nanoparticles had a mean hydrodynamic diameter of 123 nm (Table 1). The size distribution was unimodal with a polydispersity index (PDI) of 0.19 and the surface charge was positive. The FITC percentage content was found to be 3.51%
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Table 2 Particle size and surface charge of chitosan:siRNA nanoparticles, in ratio 10:1, 30:1 and 50:1 w/w (mean ± SD, n = 3).
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Hydrodynamic diameter (nm ± SD)
Polydispersity index (±SD)
Zeta potential (mV ± SD)
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+35 ± 2 +40 ± 1 +41 ± 1
(w/w) with a labelling efficiency of 91%. The size and surface charge properties of the FITC-labelled chitosan formulation was not significantly different (p > 0.05) from the corresponding unlabelled formulation (Table 1), indicating that FITC labelling did not alter these important parameters, probably due to the low degree of conjugation and low molecular weight of FITC.
Inclusion of siRNA within chitosan nanoparticle formulations at various ratios did not produce a significant difference (p > 0.05) in mean nanoparticle particle size (Table 2), with the mean particle size for all formulations being less than 140 nm. With a higher amount of siRNA, i.e. chitosan to siRNA in ratio of 10:1 (w/w), the polydispersity index was 0.22 whereas with less siRNA, the polydispersity index was smaller. The surface charge of siRNA loaded nanoparticles ranged from +35 to +41 mV (Table 2). These values were not significantly different (p > 0.05) when compared to unloaded labelled and unlabelled nanoparticles (Table 1). Comparing siRNA-loaded formulations in different ratios, in general as the siRNA loading decreased, a higher surface charge was obtained. 3.3. Scanning electron microscopy of nanoparticles
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3.4. Effect of pH on nanoparticle properties
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In order to explore the potential of crosslinked chitosan nanoparticles for pulmonary delivery, it is important to consider
6.5
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pH Fig. 2. Surface charge of crosslinked chitosan nanoparticles at a range of pH values (mean ± SD, n = 4).
their behaviour at lung pH, i.e. pH 6.5 (Mohri et al., 2010). As there is no change in size and surface charge of siRNA loaded chitosan nanoparticles for these studies unloaded chitosan nanoparticles are used for pH stability studies. The change in surface charge of nanoparticles over the pH range 5.4–8.0 is shown in Fig. 2. In acidic pH, 5.4, the nanoparticles had a maximum surface charge of +46 mV. The surface charge reduced as the pH increased. The nanoparticles were still positively charged even at a pH as high as 7.9. The change in hydrodynamic diameter of chitosan nanoparticles at different pH values (5.4–8.0) is shown in Fig. 3. Over the pH range 5.4–6.8, nanoparticle size remained constant, with mean hydrodynamic diameters less than 200 nm. The size increased to greater than 1 m when the pH of the medium was raised above 7.4, larger than can be accurately measured by dynamic light scattering. With increased pH, particle aggregation visibly occurred, leading to a turbid dispersion. 3.5. Encapsulation efficiency of nanoparticles
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Values out of range
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pH Fig. 1. Scanning electron micrograph of freeze-dried FITC-labelled crosslinked chitosan nanoparticles.
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An encapsulation efficiency of >96% was achieved for chitosan:siRNA nanoparticles in all ratios (Fig. 4). When comparing
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3.2. Size and surface charge of siRNA encapsulated nanoparticles
Following freeze-drying, SEM of fluorescent labelled particles showed spherical nanosized structures with almost uniform size distribution (Fig. 1). Similar observations were made for unlabelled nanoparticles. These fluorescent chitosan nanoparticles were used in further studies.
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Zeta potenal (mV)
Chitosan:siRNA (w/w)
Fig. 3. Influence of pH on the hydrodynamic diameter of crosslinked chitosan nanoparticles (mean ± SD, n = 4).
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0.0 Nebuliser reservoir
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Fig. 4. Encapsulation efficiency of siRNA in chitosan:siRNA nanoparticles at ratio of 10:1, 30:1 and 50:1 w/w (mean ± SD, n = 3).
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siRNA-loaded nanoparticle formulations in w/w ratios of 10:1, 30:1 and 50:1, no significant difference (p > 0.05) in encapsulation efficiency was observed.
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3.6. siRNA binding affinity with chitosan nanoparticles
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Following gel electrophoresis, negatively charged free siRNA (Lane 1) migrated towards the positive electrode (Fig. 5). For chitosan:siRNA (10:1) (Lane 2), siRNA is present in a high amount with respect to the formulation and cannot to be retained completely encapsulated within the nanoparticles in the well, and consequently the siRNA moved to the same level as the control siRNA (Lane 1). At a ratio of 30:1 (Lane 3), siRNA remained almost completely within the well, with a slight ‘smear’ away from the well. At the highest ratio of 50:1 (Lane 4), complete siRNA binding was observed, with the nanoparticles retaining siRNA within the well. This suggests that as the amount of chitosan increased in the nanoparticle, binding affinity improves. Poor retention of siRNA within crosslinked chitosan (20 kDa):siRNA nanoparticles at a weight ratio of 80:1 has been described previously (Rojanarata et al., 2008).
Fig. 6. Chitosan deposited on each stage of the TSI and remaining in the nebuliser following nebulisation of FITC-labelled chitosan nanoparticles (with and without siRNA) (mean ± SD, n = 3).
3.7. Nebulisation of unloaded and siRNA-loaded chitosan nanoparticles into the TSI In vitro analysis of the aerosol performance of nanoparticles delivered from a Pari LC Sprint jet nebuliser showed that a higher proportion of chitosan was deposited on Stage 2, than Stage 1 of the TSI, indicating suitable aerosolisation properties for lung delivery (Fig. 6). Nebulised chitosan nanoparticles had a FPF of 54 ± 11% in the TSI. siRNA-loaded chitosan nanoparticles (chitosan: siRNA ratio of 10:1 w/w) had a comparable FPF of 57.3 ± 1.9% (p > 0.05), indicating that the presence of siRNA within the system (even at a relatively high concentration) did not affect the aerosolisation behaviour of nanoparticles. Chitosan recovered from the nebuliser, and all parts of TSI, achieved a mass balance of 91 ± 7% for chitosan nanoparticle formulation and 92.9 ± 1.8% for chitosan:siRNA (10:1 w/w) nanoparticle formulation, within the European Pharmacopoeia (2008) limits of 75–125%. 3.8. Gel electrophoresis following nebulisation of siRNA-loaded nanoparticles Following aerosolisation and collection of deposited material from Stage 2 of the TSI, naked siRNA could not be detected in Lane 1 following gel electrophoresis, indicating either siRNA degradation during aerosolisation or an inability to form aerosols by nebulisation (Fig. 7). The slight band in Lane 1 was the side numbering on the gel plate present by default. Pre-nebulised, chitosan:siRNA (Lane 2) nanoparticles in the w/w ratio of 10:1 showed a bright clear band mostly moving away from the well, with slight retention within the well. This was due to the relatively high ratio of negatively charged siRNA to positively charged chitosan. These results are the same as shown in Fig. 5. Following nebulisation of chitosan:siRNA nanoparticles (Lane 3), the siRNA behaved in the gel in a similar manner to the same formulation prior to nebulisation (Lane 2). The band intensity of post-nebulised siRNA observed in gel electrophoresis was less than chitosan:siRNA nanoparticles before nebulisation, due to dilution of samples within the impinger. These findings signify stability and successful aerosolisation of siRNA when associated with the chitosan oligosaccharide nanoparticles. 3.9. In vitro assay of siRNA:chitosan nanoparticle cell viability
Fig. 5. Gel electrophoresis of siRNA and siRNA encapsulated nanoparticles. Lane 1: Naked siRNA (control); Lanes 2–4: chitosan:siRNA nanoparticles at w/w ratio of 10:1, 30:1 and 50:1 respectively.
siRNA-loaded nanoparticles at a chitosan nanoparticle concentration of 2.6 g/ml did not show cellular toxicity (H-292 cells) over a period of 24 h (Fig. 8), comparable with non-treated cells (no formulation present). At the highest chitosan nanoparticle
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Fig. 7. Gel electrophoresis of siRNA before and after nebulisation, with and without chitosan, collected from Stage 2 of the TSI; Lane 1: Naked siRNA (control) after nebulisation, Lane 2: chitosan:siRNA (10:1 w/w) before nebulisation, Lane 3: chitosan:siRNA (10:1 w/w) after nebulisation.
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Cell viability (%)
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Amount of chitosan in each well (μg/ml) Fig. 8. H-292 cell viability following exposure to nanoparticles with chitosan (encapsulated siRNA in ratio of 30:1 w/w) ranging from 2.6 to 83.3 g/ml over a 24 h incubation period (mean ± SD, n = 3).
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concentration (83 g/ml) the cell viability reduced to 85%. These observations collectively demonstrate that chitosan nanoparticles loaded with siRNA, at the chitosan concentrations and siRNA ratios studied here do not affect cell viability and may be used for in vivo applications.
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4. Discussion
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The ionic interaction between the positively charged amine groups of chitosan and the negatively charged phosphate groups of TPP generated crosslinked nanoparticles. The cationic nature of these nanoparticles makes them useful for production of complexes with negatively charged siRNA for biopharmaceutical applications and for promoting high transfection efficiency within negatively charged cells, as a result of electrostatic interactions (Howard et al., 2006; Andersen et al., 2009). The surface charge of chitosan nanoparticles is dependent on pH. As the pH of the medium in which these nanoparticles were dispersed was increased, a
greater proportion of amine groups were deprotonated resulting in a decrease in the measured positive surface charge, reducing steric hindrance between particles, resulting in particle aggregation. It was observed that the physical stability of the crosslinked chitosan nanoparticles was pH dependent, but considerable aggregation did not occur at the physiological pH of the lung; i.e. 6.5. The changes in surface charge and hydrodynamic diameter correlated well over the pH range 5.4–8, in agreement with a study of chitosanenoxaparin complexes which were stable in the pH range 3–6.5, but aggregated at higher pH (Sun et al., 2008). One of the main issues with non-viral carriers is maintaining the original particle size in biological media. These studies show that the chitosan nanoparticles were physically stable at the pH likely to be encountered at their site of deposition following nebulisation. The encapsulation efficiency of a biopharmaceutical into chitosan nanoparticles is dependent on several parameters, including the molecular weight of chitosan, the nature of the biopharmaceutical, stirring speed, formulation composition, concentration and volume ratio of TPP and type of chitosan used. The methodology employed here resulted in high encapsulation efficiency (>96%) of siRNA into crosslinked chitosan nanoparticles. In a previous study, chitosan (50 k–150 kDa) nanoparticles prepared using TPP as an ionic crosslinker, encapsulating siRNA at an N:P ratio of 30:1, showed a similar encapsulation efficiency of >95% (Dehousse et al., 2010). Gel electrophoresis was performed to assess the association (encapsulation) of siRNA within nanoparticles. A clear bright uniform band was visualised. Upon siRNA interaction with chitosan the positively charged nanoparticles retarded the movement of negatively charged siRNA towards the positive electrode. The bright band of siRNA is an indication that the siRNA nanoparticle preparation was successful, maintaining the integrity of siRNA during the stage of encapsulation. Preliminary experiments were performed to investigate both high molecular chitosan (HMC; glutamate derivative) and low molecular weight chitosan (LMC) nanoparticles as potential carriers for pulmonary delivery. It was observed that upon their nebulisation the FPF for both was not significantly different (results not shown). Since LMC was a non-modified chitosan unlike HMC which was a glutamate derivative, LMC was selected for the further experiments described here. Following nebulisation from a jet nebuliser, chitosan:siRNA (10:1 w/w) nanoparticles showed a high nanoparticle deposition in Stage 2 of the TSI (57.3 ± 1.9% FPF) and less deposition in Stage 1, with a proportion of nanoparticles remaining in the residual fluid that remains in the nebuliser following nebulisation. A 10:1 ratio was considered appropriate in this experiment, since at this ratio the siRNA was partially bound and unbound to the carrier and so might be predicted to release siRNA during nebulisation, possibly resulting in denaturation, and following delivery, which is therapeutically desirable. Aerosols reaching Stage 2 of the TSI have mass median aerodynamic diameter (MMAD) <6.4 m and are predicted to reach the peripheral region of the lung. This suggests that the formulation is suitable as a carrier for delivery to the lower respiratory tract. In order to investigate the integrity of siRNA following nebulisation, the formulation deposited in Stage 2 was studied using gel electrophoresis. Following nebulisation, naked siRNA did not show any band, whilst nebulised siRNA encapsulated within nanoparticles showed a siRNA band on the gel. The stability of biopharmaceuticals may be compromised during jet nebulisation as a result of shearing forces in the nebuliser and surface effects at the liquid droplet/air interface (Niven and Brain, 1994). Association with the nanoparticles has ensured the integrity of siRNA is maintained during nebulisation. Previously, complexation of siRNA with positively charged PEI was found to be necessary to maintain stability during nebulisation (Huth et al., 2006). These findings, thus, confirm successful siRNA aerosolisation using a nebuliser and delivery to Stage
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2 of the TSI using a new class of chitosan nanoparticle formulation, indicating its potential for in vivo studies. Nanoparticles delivered to the lungs come into contact with epithelial cells lining the respiratory tract. One of the main concerns with non-viral vectors is toxicity and hence it is essential to determine the cytotoxicity of the siRNA-nanoparticle formulation towards the target site. Nanoparticles prepared at chitosan:siRNA w/w ratios of 30:1 were selected for cytotoxicity studies, because at this ratio particles showed almost complete binding to the siRNA, had an appropriate size and positive surface charge. These nanoparticles had no overt toxicity to H-292 cells following exposure at chitosan concentration of up to 83.3 g/ml (24 h). Previously, chitosan has been shown to exhibit molecular weight-dependent cytotoxic effects, with water soluble chitosan (<10 kDa) at a low concentration (<1 mg/ml) showing negligible cytotoxic effect on the Caco-2 cells (incubated over 2 h) (Chae et al., 2005). The use of chitosan in the delivery of nucleic acids is well established (Lee et al., 2001; Lai and Lin, 2009). The results obtained here are encouraging with respect to the development of crosslinked chitosan nanoparticles for pulmonary administration of siRNA. The formulation possesses suitable aerodynamic characteristics, physical stability, the ability to encapsulate siRNA and in vitro biocompatibility, as demonstrated previously (Mwale et al., 2005). 5. Conclusion
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Nebulisation of biopharmaceuticals in solution may result in their degradation. Their stability can be improved by association or complexation with carrier particles. In this study, siRNA was successfully associated with non-toxic, crosslinked chitosan nanoparticles of appropriate size and charge, which protected the siRNA against the physical stresses encountered during nebulisation The combined features of nanoparticle stability at the pH of lung airways, high encapsulation efficiency, low cytotoxicity and efficient aerosolisation suggest these biodegradable chitosan nanoparticles would be a suitable siRNA carrier for future in vivo studies.
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Acknowledgements
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Financial support from AstraZeneca is gratefully acknowledged. We also thank David McCarthy, Hamid Ali Merchant and Jonathan David Curry of UCL School of Pharmacy for technical advice.
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