Gene Silencing in a Mouse Lung Metastasis Model by an Inhalable Dry Small Interfering RNA Powder Prepared Using the Supercritical Carbon Dioxide Technique

Abstract

In this study, a novel dry small interfering RNA (siRNA) powder for inhalation, containing chitosan and mannitol, was prepared using the supercritical carbon dioxide (CO₂) technique. Although the siRNA/chitosan powder was difficult to disperse because of a long needle-like structure, it could be reduced to fragments of 10–20 µm by manual grinding, which allowed for administration into mice. Electrophoresis revealed that the supercritical CO₂ technique and manual grinding didn’t greatly affect the integrity of the siRNA. Furthermore, the siRNA was more stable in the lungs than in blood, suggesting the utility of pulmonary delivery. Biodistribution experiments using Cy5.5-labeled siRNA demonstrated that pulmonary administration of the powder achieved a prolonged exposure of the siRNA/chitosan complex on the lung epithelial surface at a higher concentration. For the evaluation of the in-vivo gene silencing effect of the siRNA/chitosan powder, mice bearing colon26/Luc cells were used. The powder significantly inhibited the increase in luminescence intensity in the lungs, but the siRNA/chitosan solution and a non-specific dry siRNA/chitosan powder didn't, indicating the effective and specific gene silencing against the tumor cells metastasized in the lungs of mice by the siRNA/chitosan powder. These results strongly indicate that inhalable dry siRNA powders have the possibility of effective pulmonary gene silencing and that the supercritical CO₂ technique can be applied to the production.

RNA interference (RNAi), first reported by Fire et al., is a unique cellular phenomenon in which double-stranded RNA (dsRNA) regulates gene expression, contributing to cellular defenses against viral infections and transposon expansion.¹⁾ Synthetic small interfering RNA (siRNA), consisting of 21–23 nucleotides, can interfere with the expression of specific genes in mammalian cells, and has been applied to gene functional analyses. In addition, siRNA has potential applications against several intractable and lethal diseases, and clinical trials against wet age-related macular degeneration, diabetic macular edema, and cancer have already started.²⁾

Key to the successful clinical application of siRNA is an efficient delivery system, overcoming both physicochemical and biological obstacles.³⁾ The polyanionic nature of siRNA limits cellular uptake via endocytosis since electrostatic repulsion to the negatively charged cellular membranes can not be avoided. Furthermore, the macromolecular character of siRNA restricts its passive diffusion through cell membranes. The systemic administration of siRNA has been performed for in-vivo studies, but a high dose of siRNA is required due to a short half-life and poor targeting ability. In pharmacokinetic studies of naked siRNA following intravenous administration, van de Water et al. and Santel et al. found that the siRNA accumulated mainly in the kidneys and was excreted in urine.^4,5) Furthermore, naked siRNA is rapidly degraded by endonucleases in the presence of serum.⁶⁾ On the other hand, non-targeted siRNA might cause severe side effects including production of interferon.⁷⁾ For efficient siRNA delivery, several pharmaceutical approaches including chemical modifications and the application of vectors have been tried, and the local administration of siRNA has preceded in clinical trials.³⁾

Recently, the pulmonary administration of siRNA has been actively carried out for efficient gene silencing in the lungs.^8–12) The approach enables the direct delivery of siRNA deep into the lungs through the respiratory tract. For tests using small animals, the intranasal or intratracheal instillation of sample solutions has often been performed. Zhang et al. demonstrated that the intranasal instillation of naked heme oxgenase-1 (HO-1) siRNA had significant lung-specific action through apoptosis in mouse lung during ischemia-reperfusion.⁸⁾ In addition, Howard et al. reported that the intranasal instillation of siRNA specific to enhanced green fluorescent protein (EGFP) gene/chitosan nanoparticles decreased the number of EGFP-expressing endothelial cells of the bronchioles in transgenic EGFP-expressing mice.¹¹⁾ Thus, pulmonary administration of siRNA has been desired as a new therapeutic treatment for lung diseases including viral infections, cancer, and cystic fibrosis.¹²⁾ On the other hand, the development of inhalable siRNA formulations is essential for clinical use.

At present, three aerosol inhalation systems are available for clinical application; nebulizers, pressurized metered-dose inhalers (pMDIs), and dry powder inhalers (DPIs).¹³⁾ A nebulizer formulation of anti-respiratory syncytial virus (RSV) siRNA (ALN-RSV01) has already been developed by Alnylam Pharmaceuticals and a phase II clinical trial is now in progress (www.anylam.com). Among these systems, however, DPIs have generated great interest due to their low cost, portability, lack of propellants, and ease of handling.¹³⁾ For the preparation of inhalable dry powders, several methods such as milling, spray-drying (SD), and lyophilization have been applied.¹⁴⁾ However, physical stress such as heating, freezing, spraying, and shear force during the preparation might have a critical effect on the siRNA or delivery system. Jensen et al. first reported the stable preparation of an inhalable dry siRNA powder containing poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles at ambient temperature.¹⁵⁾ On the other hand, Yadava et al. clarified that lyophilization dramatically changed the particle size of a siRNA/cationic liposome complex, resulting in a loss of transfection efficiency.¹⁶⁾ Therefore, a reliable method of preparing inhalable dry siRNA powders is necessary to achieve efficient pulmonary siRNA therapy.

The supercritical fluid technique is an alternative approach for several dry powder formulations including sugars, steroids, biodegradable microspheres, and liposomes.^17–21) In particular, supercritical carbon dioxide (CO₂) has been commonly used as a solvent or antisolvent because of an easily accessible critical point (31.1°C, 7.38 MPa), non-oxidation, low cost, non-flammability, environmental acceptance, and ease of recycling.²²⁾ These characteristics have greatly contributed to the production of powders of several proteins such as insulin, lysozyme, and catalase without losses of activity.^23,24) However, there have been few reports about the application of supercritical CO₂ to powders of nucleic acids including siRNA for inhalable gene delivery. In our and the previous reports, dry plasmid DNA (pDNA) powders could be stably prepared by the supercritical CO₂ technique.^25–29) We demonstrated that a pDNA powder prepared with chitosan, a biodegradable cationic polymer, exhibited a greater pulmonary gene expressing effect and longer storage than did the solution.^26,27) These observations prompted us to use the supercritical CO₂ technique to prepare inhalable siRNA powders.

Therefore, in this study, an inhalable dry siRNA/chitosan powder was prepared by the supercritical CO₂ technique. The morphology of the powder, the stability of the siRNA during preparation of the powder, and the physicochemical characteristics of the siRNA/chitosan complex were evaluated. Furthermore, the biodistribution and gene silencing effect of siRNA were investigated following pulmonary administration of the powder into mice.

Materials and Methods

Materials

The siRNA specific to the firefly luciferase gene (siGL3: sense; 5′-CUUACGCUGAGUACUUCGAdTdT-3′, antisense; 5′-UCGAAGUACUCAGCGUAAGdTdT-3′) and that specific to the renilla luciferase gene (siRL: sense; 5′-AAACAUGCAGAAAAUGCUGdTdT-3′, antisense; 5′-CAGCAUUUUCUGCAUGUUUdTdT-3′) were purchased from Samchully Pharm. Co., Ltd., Seoul, Korea. siRL was selected as a control siRNA non-specific to the firefly luciferase gene. Cy5.5-siGL3 was synthesized by the conjugation of Cy5.5 to the 5′ end of the antisense chain in siGL3 (Samchully Pharm. Co., Ltd.). Chitosan (molecular weight (M_w); 2000–5000, water-soluble) and mannitol (Wako Pure Chemical Industries, Ltd., Osaka, Japan) were used as a non-viral vector and an excipient, respectively. Luciferin, a substrate of firefly luciferase, was obtained from Promega Co. (Madison, U.S.A.). Isoflurane, an inhalable anesthetic, was purchased from Abbott Laboratories (Abbott Park, U.S.A.). The other reagents and solvents used were of analytical grade.

Cells

Murine colon adenocarcinoma cells carrying the firefly luciferase gene (colon26/Luc cells) were kindly provided by Prof. Y. Takakura, Kyoto University. They were cultured in RPMI1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 100 units/mL penicillin, and 100 µg/mL streptomycin at 37°C in humidified air containing 5% CO₂.

Animals

BALB/c mice (male, 4 or 6 weeks old) were purchased from Japan SLC, Inc. (Shizuoka, Japan). All animal experiments were carried out in accordance with the Guiding Principles for the Care and Use of Laboratory Animals approved by the Faculty of Pharmacy, Meijo University.

Preparation of the siRNA/Chitosan Powder by the Supercritical CO₂ Technique

A dry siRNA/chitosan powder was prepared, based on the supercritical antisolvent method, as described in our previous reports.^26–29) The apparatus for preparing the powders was assembled by JASCO Co., Tokyo, Japan and composed of pumps (for CO₂, ethanol, and water), an oven, and a back pressure regulator. In brief, all components (total mass; 50 mg), as listed in Table 1, were dissolved in 1 mL of water. The solution was injected into the water flow through a manual injector. Flow rates of CO₂, ethanol, and water were set to 14 mL/min, 0.665 mL/min, and 0.035 mL/min, respectively. The three solvents were mixed in a compressed column (35°C, 25 MPa) to precipitate the components of samples. At 90 min after sample injection, the flows of water and ethanol were stopped and that of CO₂ was continued for an additional 60 min to completely remove the water and ethanol in the column. Following the release of pressure, the dry powder was collected from the column. The powder was ground for 5 min manually using a pestle and mortar to improve its dispersibility.

Table 1. Composition of Dry siRNA Powders (DPs) and siRNA Solutions (SLs) (Dosage/Body)

Dry powder (DP) formulations
Formulation name	N/P	siGL3 (µg)	siRL (µg)	Cy5.5-siGL3 (µg)	Ch (µg)	Man (µg)	Total mass (µg)
siGL3/Ch DP	10	60	—	—	300	2640	3000
siRL/Ch DP	10	—	60	—	300	2640	3000
Cy5.5-siGL3/Ch DP	10	—	—	60	300	2640	3000
Solution (SL) formulations
Formulation name	N/P	siGL3 (µg)	Cy5.5-siGL3 (µg)	Ch (µg)	Water (µL)
siGL3/Ch SL	10	60	—	300	100
Naked Cy5.5-siGL3 SL	—	—	60	—	100
Cy5.5-siGL3/Ch SL	10	—	60	300	100

N/P; molecular ratio of amine in chitosan to phosphate in siRNA, Ch; chitosan, Man; mannitol.

Abbreviations of Formulation Names

The formulation names for prepared dry powders and solutions were abbreviated as “DP” and “SL” in each Figure and Table, respectively.

Morphology of the siRNA/Chitosan Powder

To examine the morphology of the siRNA/chitosan powder, the particles were observed using a scanning electron microscope (Type JSM-6060, SEM, JEOL Ltd., Tokyo, Japan).

Particle Size and Zeta Potential of the siRNA/Chitosan Complex

The particle size and zeta potential of the siRNA/chitosan complex were measured using a Zetasizer® Nano ZS (Malvern Instruments Ltd., Worcestershire, U.K.). The siRNA/chitosan powder was dissolved in water to reform the siRNA/chitosan complex. Each sample was adjusted to a siRNA concentration of 20 µg/mL.

Preparation of the Lung Homogenate

The lung homogenate was prepared as reported by Fukuda et al.³⁰⁾ BALB/c mice (6 weeks old) were anesthetized with pentobarbital (50 mg/kg, intraperitoneally (i.p.)) and sacrificed by exsanguination from the vena cava. Phosphate-buffered saline (PBS) 20 mL was infused from the right ventricle into the pulmonary artery to eliminate all blood in the lungs. The lungs were excised, and stored at −80°C. Just prior to use, the frozen lungs were thawed at room temperature, and homogenized in cold PBS using a tissue homogenizer (OMNI International Co., Kennesaw, U.S.A.). The homogenate was centrifuged at 5000×g for 10 min to collect the supernatant. The concentration of protein in the supernatant was measured using the Bradford assay, and the supernatant was adjusted with PBS to a protein concentration of 1.0 mg/mL for use as the lung homogenate.

Stability of siRNA in Serum and the Lung Homogenate

A PCI solution (phenol–chloroform–isoamyl alcohol=10 : 3.43 : 0.07, volume ratio) was centrifuged at 13000×g for 1 min to remove water, and cooled on ice until used. siGL3 was dissolved in PBS at a concentration of 1 µM. Fifteen microliters of the siGL3 solution was added to 35 µL of fetal bovine serum or lung homogenate (1.0 mg/mL as protein concentration), and the mixtures were incubated at 37°C. At various time points, 10 µL of each mixture was added to 13.5 µL of the PCI solution. After centrifugation at 4°C, 13000×g, for more than 1 h, the mixtures containing the PCI solution were separated into three phases (phenol, water, and protein) and the water phase was collected for electrophoresis.

Determination of siRNA Integrity by Electrophoresis

Each sample containing approximately 0.01 µg of siRNA and a siRNA marker (N2101S, New England Bio Labs, Inc., Ipswich, U.S.A.) were loaded onto a 15% polyacrylamide gel. Electrophoresis was carried out with a current of 250 V, 30 mA for 45 min in Tris-borate-ethylenediamine tetraacetic acid (EDTA) running buffer. After the electrophoresis, the gel was soaked in a 0.05% ethidium bromide solution for detecting siRNA with a fluorescent image analyzer (FMBIO® II Multi-View; Hitachi Software Engineering Co., Ltd., Tokyo, Japan). To examine the integrity of siRNA in pre- and post-ground dry siRNA/chitosan powders, they were dissolved in water, and the same procedure was performed.

Administration of the siRNA/Chitosan Powder

Mice were anesthetized with pentobarbital (50 mg/kg, i.p.) and a board was secured on their backs. The trachea was exposed and 2.5 cm of PE-60 polyethylene tubing (internal diameter: 0.76 mm, Becton Dickinson and Company, Franklin Lakes, U.S.A.) was inserted to a depth of 1.0 cm through an incision. The ground siRNA/chitosan powder was administered through the trachea using an appropriate apparatus for mice.³¹⁾ One and half milligrams of the powder (30 µg of siRNA) was put in a disposable tip and dispersed in the trachea by releasing air (0.25 mL) compressed in a syringe by opening a three-way stopcock connecting the disposable tip and the syringe.

Biodistibution of siRNA

The Cy5.5-siGL3/chitosan powder was administered twice a total of 60 µg of siRNA into BALB/c mice (6 weeks old) as above. However, 50 µL each of the naked Cy5.5-siGL3 solution and the Cy5.5-siGL3/chitosan solution were administered following the procedure, which had the same dose of Cy5.5-siGL3/chitosan powder as siRNA and chitosan. At various time points, the fluorescence derived from Cy5.5 in mice (Ex, 675 nm; Em, 720 nm) was detected using an in-vivo imaging system (IVIS®; IVIS-SPECTRUM, Caliper Life Sciences, Hopkinton, MA, U.S.A.). The exposure time was set to 1 s. During the measurement, the mice were anesthetized with isoflurane on a stage kept at 37°C. To measure the fluorescence intensity for lung and non-lung compartments in each mouse, the regions of interest (ROIs) were adjusted to rectangular forms with a width of 3.5 cm and height of 1.2 cm for lung compartments and a width of 3.5 cm and height of 3.6 cm for non-lung compartments, as shown in Fig. 5B. The lung fluorescence localization in each mouse was calculated as follows:

  

where F_L and F_NL were the fluorescence intensity in lung and non-lung compartments, respectively.

Lung Metastasis

Lung metastasis was established as reported previously.²⁹⁾ After being harvested with EDTA-trypsin solution, colon26/Luc cells were prepared at a concentration of 1×10⁶ cells/mL using PBS. Then, 100 µL of the cell suspension was intravenously injected into the tail of each BALB/c mouse (4 weeks old). The lung luminescence intensity corresponding to the luciferase activity of inoculated colon26/Luc cells was monitored with IVIS® at 10 min following the intraperitoneal administration of luciferin (150 mg/kg), and the exposure time was set to 1 min. During the measurement, the mice were anesthetized with isoflurane on a stage kept at 37°C. The ROI for lung luminescence intensity in each mouse was adjusted to a circular form with a diameter of 2.8 cm.

In-Vivo Gene Silencing

After the luminescence in the lungs reached 1.0–2.5×10⁵ photons/s (on day 9–14 following the inoculation), treatment was started. The siRNA/chitosan powders 1.5 mg were administered twice per day (a total of 60 µg/d of siRNA) for two days. In contrast, 50 µL of the siRNA/chitosan solution was administered following the same procedure, which had the same dose of siRNA/chitosan powder as siRNA and chitosan. The luminescence intensity in the treated mice was measured using IVIS® at each time point as mentioned above, and relative luminescence intensity was calculated as the ratio to that on day 0 after the first treatment.

Statistical Analysis

Statistical comparisons were made using Tukey’s multiple comparison test for physicochemical study of the complex and biodistribution one, and Dunnett’s multiple comparison test for pulmonary gene silencing one, respectively. A p<0.05 was considered significant.

Results

Preparation and Morphology of the siRNA/Chitosan Powder Prepared by the Supercritical CO₂ Technique

All the siRNA/chitosan powders could be successfully collected by the supercritical CO₂ technique. The recovery rate was 54.9±16.9% (mean±S.D., n=6). From S.E.M., the powder comprised long needle-like particles (Fig. 1A). Unfortunately, it could not be dispersed using an apparatus for mice,³¹⁾ which suggested that it was unsuitable for inhalation. Therefore, manual grinding was carried out to improve its dispersibility. The ground siRNA/chitosan powder had fragments approximately 10–20 µm long (Fig. 1B), which allowed for pulmonary administration into mice.

Fig. 1. Scanning Electron Micrographs of the siRNA/Chitosan Powders Prepared by the Supercritical CO₂ Technique

(A) Pre-ground siGL3/Ch DP, and (B) post-ground siGL3/Ch DP.

Integrity of the siRNA in the Powder

To investigate the integrity of the siRNA in the siRNA/chitosan powder, electrophoresis was carried out. A single band corresponding to the siRNA was detected at the position corresponding to 21 bp (Fig. 2B), indicating that the siRNA remained stable in the powder. Furthermore, a similar result was observed in the ground powder (Fig. 2C), suggesting that the manual grinding had no effect on siRNA integrity in the siRNA/chitosan powder.

Fig. 2. Integrity of siRNA in the siRNA/Chitosan Powders Prepared by the Supercritical CO₂ Technique

Physicochemical Properties of the siRNA/Chitosan Complex

To evaluate the physicochemical properties of the siRNA/chitosan complex before and after the powder was produced, the particle size and zeta potential of the complex were measured (Fig. 3). There was no dramatic change in either parameter before and after the powder was produced, with values of approximately 130 nm and −21 mV, respectively. Additionally, similar results were obtained with the ground powder, and with the Cy5.5-siRNA/chitosan complex, although small but statistically significant differences were detected in the Cy5.5-siRNA/chitosan complex. Therefore, the supercritical CO₂ technique and manual grinding did not cause a marked change in the physicochemical properties of the siRNA/chitosan complex.

Fig. 3. (A) Particle Size and (B) Zeta Potential of the siRNA/Chitosan Complex before and after the Powder Was Produced by the Supercritical CO₂ Technique

Pre- and post-ground powders were dissolved in water to reform the siRNA/chitosan complex. Each value represents the mean±S.D. (n=3). Statistically significant differences compared with before (** p<0.01) and after (^† p<0.05).

Stability of siRNA in Lung

From electrophoresis, the band corresponding to the siRNA was shifted from the position of 21 to 17 bp as time passed after the mixing with serum (Fig. 4), suggesting a rapid degradation of the siRNA in serum. For 2 h after the siRNA and lung homogenate were mixed, on the other hand, no shift was observed from the 21-bp position, indicating the stability of siRNA in lung. It was confirmed that some bands above the lane were derived from the lung homogenate.

Fig. 4. Stability of siRNA in Serum and Lung Homogenate

Biodistribution of siRNA Following Pulmonary Delivery of the siRNA/Chitosan Powder

To investigate the biodistribution of siRNA following pulmonary administration, Cy5.5-siRNA was applied and the fluorescence derived from Cy5.5 in mice was detected using an in-vivo imaging system. An optical biodistribution image of siRNA is shown in Fig. 5A. One hour following the pulmonary delivery of the naked Cy5.5-siRNA solution into mice, fluorescence derived from Cy5.5 was detected not only in the lung but also in the liver, suggesting the absorption of siRNA from the lung into the systemic circulation. Subsequently, fluorescence was also detected in the intestine at 6 h, which might show the parts transferred by biliary excretion or that swallowed following mucociliary clearance. On the other hand, the translocation from the lung to liver was delayed using both the Cy5.5-siRNA/chitosan solution and the Cy5.5-siRNA/chitosan powder. These results strongly indicated that chitosan prolonged the retention of siRNA in the lungs.

Fig. 5. Biodistribution of siRNA Following Pulmonary Delivery of the siRNA/Chitosan Powder into Mice

(A) Optical image of the fluorescence derived from Cy5.5 in mice following pulmonary delivery of naked Cy5.5-siGL3 SL, Cy5.5-siGL3/Ch SL, and Cy5.5-siGL3/Ch DP into mice. The fluorescence derived from Cy5.5 in mice was detected using IVIS®. The color scales are in photons/s/cm²/sr. (B) Lung and non-lung compartments in each mouse. The regions of interest (ROIs) were adjusted to rectangular forms with a width of 3.5 cm and height of 1.2 cm for the lung compartment and with a width of 3.5 cm and height of 3.6 cm for the non-lung compartment. These sizes were determined from a dissected mouse. (C) Time course of fluorescence intensity derived from Cy5.5 in the lung compartment following pulmonary delivery of naked Cy5.5-siGL3 SL, Cy5.5-siGL3/Ch SL, and Cy5.5-siGL3/Ch DP into mice. (D) Time course of fluorescence intensity derived from Cy5.5 in the non-lung compartment following pulmonary delivery of naked Cy5.5-siGL3 SL, Cy5.5-siGL3/Ch SL, and Cy5.5-siGL3/Ch DP into mice. (E) Time course of lung fluorescence localization derived from Cy5.5 following pulmonary delivery of naked Cy5.5-siGL3 SL, Cy5.5-siGL3/Ch SL, and Cy5.5-siGL3/Ch DP into mice. Each value represents the mean±S.D. (n=3). Statistically significant differences compared with naked Cy5.5-siGL3 SL (** p<0.01; * p<0.05), and with Cy5.5-siGL3/Ch SL (^‡ p<0.01; ^† p<0.05).

To further examine the biodistribution, the mean fluorescence intensity in lung and non-lung compartments, shown in Fig. 5B, from three mice treated with each formulation was calculated (Figs. 5C, D). Although preliminary experiments confirmed that the fluorescence intensity of Cy5.5-siRNA decreased approximately 65% with the formation of a complex with chitosan in water (data not shown), that in the lung compartment was higher for the Cy5.5-siRNA/chitosan powder than the Cy5.5-siRNA/chitosan solution (Fig. 5C). This result indicated that a higher concentration of the siRNA/chitosan complex formed on the lung epithelial surface after the dissolution of the powder in the lungs. Moreover, it might partly explain why the fluorescence intensity in the non-lung compartment was higher for 0.5 h following the pulmonary delivery of the Cy5.5-siRNA/chitosan powder (Fig. 5D). From 1 h after the pulmonary administration, on the other hand, both the solution and powder tended to exhibit less fluorescence in the non-lung compartment than did the naked Cy5.5-siRNA solution, suggesting a prolonged pulmonary retention of the siRNA.

Unfortunately, there was relatively extensive dispersion of the fluorescence in lung and non-lung compartments among mice treated with each formulation. This was considered due to difficulty with the pulmonary delivery of each formulation at an exact dose because of the influences of spontaneous breathing and mucociliary clearance backflow. To compensate, therefore, lung fluorescence localization was calculated from the fluorescence intensity in lung and non-lung compartments in each mouse (Fig. 5E). From this evaluation, the prolonged retention of Cy5.5-siRNA in the Cy5.5-siRNA/chitosan solution and Cy5.5-siRNA/chitosan powder was clearly confirmed from 1 h following the pulmonary administration.

In-Vivo Gene Silencing by the siRNA/Chitosan Powder

For the in-vivo gene silencing experiment, mice bearing colon26/Luc cells were subjected to imaging in vivo. The luminescence in the lungs increased markedly with time, corresponding to tumor growth (Fig. 6). On days 2 and 3 after the first treatment, the siGL3 (siRNA specifically recognizing the firefly luciferase gene)/chitosan powder significantly inhibited the increase in luminescence in the lungs compared with no treatment, while the siRL (not specific to the firefly luciferase gene)/chitosan powder and siGL3/chitosan solution did not. These results strongly indicated that the siRNA/chitosan powder exhibited the effective and specific gene silencing against the tumor cells metastasized in the lungs of mice.

Fig. 6. In-Vivo Gene Silencing in Mice by the siRNA/Chitosan Powder

(A) Optical image of lung luminescence corresponding to firefly luciferase activity in mice bearing colon26/Luc cells for 3 d after the first treatment with siGL3/Ch SL, siGL3/Ch DP, and siRL/Ch DP. Colon26/Luc cells were intravenously injected into the tail. The luminescence corresponding to firefly luciferase activity was detected using IVIS®. After the luminescence intensity in the lungs reached 1.0–2.5×10⁵ photons/s (on day 9–14 following the inoculation), each treatment was started. siGL3/Ch SL, siGL3/Ch DP, and siRL/Ch DP were intratracheally injected at a dose of 60 µg/day as siRNA for 2 d, respectively. The color scales are in photons/s/cm²/sr. (B) Time course of lung luminescence intensity corresponding to firefly luciferase activity in mice bearing colon26/Luc cells for 3 d after the first treatment with siGL3/Ch SL, siGL3/Ch DP, and siRL/Ch DP. Each value represents the mean±S.D. (n=3–5). Statistically significant differences compared with N.T. (** p<0.01; * p<0.05). N.T.; no treatment.

Discussion

In this study, we used a supercritical CO₂ technique to prepare an inhalable dry siRNA/chitosan powder, and demonstrated a powerful and specific in-vivo gene silencing effect following pulmonary administration into mice. To our knowledge, this is the first report about in-vivo gene silencing by an inhalable dry siRNA powder.

Preparing a powder without a loss of siRNA integrity is essential for effective pulmonary gene silencing by siRNA DPI. Preliminary experiments revealed that no electrophoretic band corresponding to siRNA could be detected in the siRNA powder prepared without chitosan (data not shown), suggesting that the supercritical CO₂ technique caused the destabilization of siRNA. Tservistas et al. clarified that the acidic conditions caused by supercritical CO₂ with water destabilized pDNA during production of the powder.²⁵⁾ Thus, similar destabilization may have occurred to siRNA in our study. On the other hand, the addition of chitosan could improve siRNA integrity in the powder (Fig. 2). A stabilizing effect of chitosan was also observed in our previous study of a pDNA powder.²⁶⁾ The pK_a of chitosan is approximately 6.5 and protonated amines under acidic conditions electrostatically interact with anionic nucleic acids to form a complex.³²⁾ Therefore, the buffering function and the complex formed by chitosan would greatly contribute to the stabilizing effect on siRNA in powder produced by the supercritical CO₂ technique.

Besides the loss of siRNA integrity, the change in the physicochemical characteristics of the complex is also an important concern for maintaining transfection efficiency. In this study, there was no dramatic change in either the particle size or zeta potential of the complex before and after the powder was produced (Fig. 3). Although further study is needed to clarify why the physicochemical properties of the complex were preserved in this study, the relatively weak electrostatic interaction between chitosan and nucleic acids might generate conformational flexibility. For detecting the electrophoretic bands corresponding to nucleic acids, polyanions such as polyaspartic acid and heparin are generally needed to unpack nucleic acids from nucleic acid/cationic vector complexes.^33,34) Without a polyanion in this study, however, the band corresponding to siRNA could be detected in the siRNA/chitosan complex reformed after dissolution of the siRNA/chitosan powder (Fig. 2), indicating the relatively weak electrostatic interaction between chitosan and siRNA compared with other cationic vectors such as polyethyleneimine. Additionally, the particle size and zeta potential of the pDNA/chitosan complex were maintained through the supercritical CO₂ and spray-freeze-drying techniques (data not shown), supporting our hypothesis in part.

For achieving effective gene silencing by siRNA in vivo, it is important to understand the biodistribution and pharmacokinetics of siRNA. It has been clarified that naked siRNA is unstable in blood because of degradation by endonucleases and that when injected intravenously, siRNA is mostly accumulated in the kidneys and excreted in urine.^4–6) However, there are few reports about the biodistribution and pharmacokinetics of siRNA following pulmonary administration. In this study, we investigated the fluorescence derived from Cy5.5-siRNA in vivo. Following the pulmonary administration of naked Cy5.5-siRNA into mice, the fluorescence transferred from the lung to the liver and intestine (Fig. 5A). The results of electrophoresis demonstrated that siRNA could stably exist in the lungs (Fig. 4). Similarly, we examined the stability of Cy5.5-siRNA in lung homogenate by electrophoresis. The fluorescence derived from Cy5.5 was detected at the position of Cy5.5-siRNA, suggesting that Cy5.5 could stably bind to the end of siRNA in lung (data not shown). These results indicate that a large part of the Cy5.5-siRNA detected in the lung compartment would have kept its integrity. On the other hand, the fluorescence detected in the non-lung compartment would be derived from not only Cy5.5-siRNA but also the parts degraded by endonucleases or free Cy5.5. In a pharmacokinetic study of radiolabeled naked siRNA following pulmonary administration into mice, Merkel et al. found that approximately 1%/mL of siRNA was detected in blood, supporting our results in part.³⁵⁾ In general, siRNA has a relatively large molecular weight and net negative charge, which limits its ability to permeate membranes.³⁾ However, macromolecules are more effectively absorbed from the lung than from other organs including the small intestine.³⁶⁾ The siRNA and Cy5.5-siRNA used in this study had a molecular weight of approximately 13–14 kDa, small enough to be absorbed intact via the lungs.

In this study, lung metastasis mice bearing CT26/Luc cells were established to evaluate the in-vivo gene silencing effect of siRNA in the lungs following pulmonary administration. Al-Mehdi et al. reported that the tumor cells metastasized in the lungs could penetrate the vascular wall by pseudopodium to extend beyond the endothelium into the alveolar space.³⁷⁾ In our previous study, moreover, it was demonstrated that the gene expressing efficiency against metastasized tumor cells was similar with that against normal cells in the lungs following pulmonary delivery of a dry pDNA/chitosan powder into lung metastasis mice.²⁹⁾ These observations strongly supported that inhalable genes could access tumor cells metastasized in the lungs. Thus, it was considered that lung metastasis mice bearing tumor cells stably expressing a reporter gene could be used as an in-vivo experiment model for inhalable siRNA formulations.

Chitosan is a promising non-viral vector for the delivery of siRNA because of its biodegradability and tolerability in the body.³⁸⁾ Howard et al. reported the pulmonary gene silencing effect of a siRNA/chitosan complex in mice following the intranasal administration of an aqueous formulation.¹¹⁾ In the present study, however, the siRNA/chitosan solution did not show an in-vivo gene silencing effect following pulmonary administration (Fig. 6). The discrepancy could be explained by the different physicochemical properties of the siRNA/chitosan complex. The molecular weight and degree of deacetylation of chitosan greatly affect its electrostatic interaction with nucleic acids, closely related with the physicochemical properties of the complex.^39–41) In the report by Howard et al., a chitosan with a high molecular weight (170 kDa) and extensive deacetylation (84%) was used, which was soluble in acidic solution but not in water.¹¹⁾ On the other hand, we selected a water-soluble chitosan of a low molecular weight (2–5 kDa) and deacetylation (40–50%) for clinical application. Moreover, the N/P ratio in vivo was 6 in the report by Howard et al.¹¹⁾ and 10 in the present study. Thus, the physicochemical properties of siRNA/chitosan complexes differed greatly between the study by Howard et al. and the present study; the particle size and zeta potential of the complex were approximately 220 nm and +19 mV in acetate buffer at pH 5.5, respectively,¹¹⁾ while those in the present study were approximately 130 nm and −21 mV in water, respectively. The positive surface charge of the complex can electrostatistically interact with negatively charged cellular membranes, leading to effective internalization into cells via endocytosis.⁴²⁾ Therefore, the complex in the present study had little potential to be internalized into cells due to its negative surface charge, resulting in a low gene silencing efficacy with our aqueous formulation. This was also consistent with the prolonged lung retention by Cy5.5-siRNA/chitosan solution compared with naked Cy5.5-siRNA solution, as shown in Fig. 5, which would mean the low internalization into lung epithelial cells and the low membrane permeation in lung. Moreover, in regard to the optimized form of chitosan for in-vitro siRNA transfection, Liu et al. reported that a high molecular weight (64.8–170 kDa), degree of deacetylation (ca. 80%), and N/P ratio (150) were more effective,⁴¹⁾ partly supporting the low transfection efficiency of the siRNA/chitosan complex in our aqueous formulation.

Interestingly, the siRNA/chitosan powder exhibited effective and specific gene silencing against the tumor cells metastasized in the lungs of mice (Fig. 6), which greatly expected us the possibility of pulmonary gene silencing by the siRNA/chitosan powder. Enhanced pulmonary transfection was also observed using a dry pDNA/chitosan powder, as shown in our previous reports.^26,29) Although it is still unclear why the siRNA/chitosan powder had a great in-vivo gene silencing effect than did the siRNA/chitosan solution, the difference is likely related to the biodistribution of siRNA (Fig. 5). After reaching the lungs, the siRNA/chitosan powder was easily dissolved in a small volume of water on the lung epithelial surface to form a higher concentration of the siRNA/chitosan complex. This is greatly supported by the results shown in Fig. 5C, where the fluorescence intensity in the lung compartment derived from Cy5.5 was higher following pulmonary delivery of the Cy5.5-siRNA/chitosan powder than the Cy5.5-siRNA/chitosan solution. Consequently, the siRNA/chitosan complex should be rapidly taken up into the lung epithelial cells, which was supposed by the higher fluorescence intensity derived from Cy5.5 in the non-lung compartment following pulmonary delivery of the Cy5.5-siRNA/chitosan powder (Fig. 5D). Since the siRNA/chitosan complex was spread on the lung epithelial surface to lower the concentration as time passed, the prolonged retention of siRNA would occur from 1 h after the administration of the siRNA/chitosan powder, similar to the siRNA/chitosan solution (Fig. 5E). From these results, it was hypothesized that the initial rapid uptake of siRNA into the lung epithelial cells, but not the prolonged retention, contributes to the effective in-vivo gene silencing by the siRNA/chitosan powder.

The morphology and particle size of a powder are critical factors affecting its inhalation. In general, aerodynamic particles of 1–5 µm are considered suitable for delivery by inhalation.⁴³⁾ On the other hand, particles of similar geometric size generate a strong adhesion force, resulting in poor dispersibility.⁴³⁾ In this study, the siRNA/chitosan powder produced by the supercritical CO₂ technique had long needle-like particles (Fig. 1A), and was hard to disperse. In our previous report, it was revealed that a dry pDNA/chitosan powder prepared by the supercritical CO₂ technique had a rectangular shape, with a short length <10 µm and long length >10 µm, and could be easily dispersed.²⁶⁾ The discrepancy was considered to be caused by the difference in composition (2% siRNA and 10% chitosan vs. 0.2% pDNA and 0.94% chitosan (of total mass)), or the difference between base pair numbers of nucleic acids (siRNA; 21 bp, pDNA; 7.1 kbp). To improve the dispersibility of the siRNA/chitosan powder, manual grinding was carried out, reconstructing fragments 10–20 µm in length (Fig. 1B). We are developing one-step methods for preparing inhalable dry siRNA/chitosan powders by supercritical CO₂ or other techniques. In our previous report, a low-density dry pDNA/chitosan powder, containing lactose as an excipient, with a sea urchin-like shape, was prepared by the supercritical CO₂ technique.²⁸⁾ As an alternative one-step method for inhalable dry siRNA/chitosan powders, a spray-freeze-drying technique might be applied since a porous dry pDNA/chitosan powder could be stably prepared as shown in our previous report.⁴⁴⁾

Acknowledgment

We thank Prof. Y. Takakura, Kyoto University, for providing colon26/Luc cells.

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