July 2013 1183Biol. Pharm. Bull. 36(7) 1183–1191 (2013)

© 2013 The Pharmaceutical Society of Japan

Regular Article

Gene Silencing in a Mouse Lung Metastasis Model by an Inhalable Dry Small Interfering RNA Powder Prepared Using the Supercritical Carbon Dioxide TechniqueTomoyuki Okuda, Daisuke Kito, Ai Oiwa, Michiko Fukushima, Daiki Hira, and Hirokazu Okamoto*Faculty of Pharmacy, Meijo University; 150 Yagotoyama, Tempaku-ku, Nagoya 468–8503, Japan.Received February 25, 2013; accepted April 6, 2013

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. Further-more, 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.

Key words small interferingRNA; inhalabledrypowder; supercriticalfluid technique;pulmonarygenede-livery; chitosan

RNA interference (RNAi),first reportedbyFireet 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,caninterferewiththeexpressionofspecificgenesin 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 macu-lar edema, and cancer have already started.2)

Key to the successful clinical application of siRNA is an ef-ficientdeliverysystem,overcomingbothphysicochemicalandbiological 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 stud-ies,but ahighdoseof siRNA is requireddue to a shorthalf-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 accumu-lated mainly in the kidneys and was excreted in urine.4,5) Fur-thermore, 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 pharma-ceutical approaches including chemicalmodifications and theapplication of vectors have been tried, and the local adminis-

tration of siRNA has preceded in clinical trials.3)

Recently, the pulmonary administration of siRNA has been actively carried out for efficient gene silencing in thelungs.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 instilla-tion of sample solutions has often been performed. Zhang et al. demonstrated that the intranasal instillation of naked heme oxgenase-1 (HO-1) siRNAhad significant lung-specificactionthrough apoptosis in mouse lung during ischemia-reperfu-sion.8) In addition, Howard et al. reported that the intranasal instillation of siRNA specific to enhanced green fluorescentprotein (EGFP) gene/chitosan nanoparticles decreased the number of EGFP-expressing endothelial cells of the bronchi-oles in transgenic EGFP-expressing mice.11) Thus, pulmonary administration of siRNA has been desired as a new therapeu-tic treatment for lung diseases including viral infections, can-cer, and cysticfibrosis.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, how-ever, 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

* To whom correspondence should be addressed. e-mail: okamotoh@meijo-u.ac.jp

Theauthorsdeclarenoconflictofinterest.

Highlighted Paper selected by Editor-in-Chief

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critical effect on the siRNA or delivery system. Jensen et al. firstreportedthestablepreparationofaninhalabledrysiRNApowder containing poly(d,l-lactide-co-glycolide) (PLGA) nanoparticles at ambient temperature.15) On the other hand, Yadava et al.clarifiedthatlyophilizationdramaticallychangedthe particle size of a siRNA/cationic liposome complex, result-ing in a loss of transfection efficiency.16) Therefore, a reliable method of preparing inhalable dry siRNA powders is neces-sarytoachieveefficientpulmonarysiRNAtherapy.The supercritical fluid technique is an alternative approach

for several dry powder formulations including sugars, ste-roids, biodegradable microspheres, and liposomes.17–21) In particular, supercritical carbon dioxide (CO2) has been com-monly 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 easeof recycling.22) These characteristics have greatly contributed to the production of powders of several proteins such as in-sulin, 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 cat-ionic polymer, exhibited a greater pulmonary gene expressing effect and longer storage than did the solution.26,27) These ob-servations prompted us to use the supercritical CO2 techniqueto prepare inhalable siRNA powders.

Therefore, in this study, an inhalable dry siRNA/chitosan powder was prepared by the supercritical CO2 technique.Themorphology of the powder, the stability of the siRNA during preparation of the powder, and the physicochemical character-istics of the siRNA/chitosan complex were evaluated. Further-more, 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 luciferasegene (siGL3: sense; 5′-CUUACGCUGAGUACUUCGAdT-dT- 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 lucif-erase gene. Cy5.5-siGL3 was synthesized by the conjugation of Cy5.5 to the 5′ end of the antisense chain in siGL3 (Sam-chully Pharm. Co., Ltd.). Chitosan (molecular weight (Mw); 2000–5000, water-soluble) and mannitol (Wako Pure Chemi-cal 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 purchasedfrom 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 pro-vided 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 containing5% 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 ap-proved by the Faculty of Pharmacy, Meijo University.

Preparation of the siRNA/Chitosan Powder by the Su-percritical 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 compo-nents (total mass; 50 mg), as listed in Table 1, were dissolved in1mLofwater.Thesolutionwasinjectedintothewaterflowthrough 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 com-pressed column (35°C, 25 MPa) to precipitate the components of samples. At 90min after sample injection, the flows ofwater and ethanol were stopped and that of CO2 was contin-ued 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.

Abbreviations of Formulation Names The formulation names for prepared dry powders and solutions were abbrevi-ated 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 homog-enate was prepared as reported by Fukuda et al.30) BALB/c mice (6 weeks old) were anesthetized with pentobarbital (50mg/kg, intraperitoneally (i.p.)) and sacrificed by exsangui-nation 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 concen-tration 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 Homog-enate A PCI solution (phenol–chloroform–isoamyl alcohol=

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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 con-centration), and the mixtures were incubated at 37°C. At vari-ous 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., Ip-swich, 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 siRNAwith 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 pow-der 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 andchitosan.Atvarious timepoints, thefluorescencederivedfrom 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 expo-sure 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-lungcompartments 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.Thelungfluorescencelocalizationineachmousewascalculated as follows:

L L NLlung fluorescence localization / ( )= +F F F where FL and FNLwere thefluorescence intensity in lungandnon-lung compartments, respectively.

Lung Metastasis Lung metastasis was established as re-ported 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 suspen-sion was intravenously injected into the tail of each BALB/c mouse (4 weeks old). The lung luminescence intensity cor-responding 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, themicewere anesthetizedwith isoflurane on a stage kept at37°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 pow-der 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 calculatedastheratiotothatonday0afterthefirsttreatment.

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<0.05wasconsideredsignificant.

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 3000siRL/Ch DP 10 — 60 — 300 2640 3000Cy5.5-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 100Naked Cy5.5-siGL3 SL — — 60 — 100Cy5.5-siGL3/Ch SL 10 — 60 300 100N/P; molecular ratio of amine in chitosan to phosphate in siRNA, Ch; chitosan, Man; mannitol.

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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 was54.9± 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 approxi-mately 10–20 µm long (Fig. 1B), which allowed for pulmonary administration into mice.

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 grind-ing had no effect on siRNA integrity in the siRNA/chitosan powder.

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, respec-tively. Additionally, similar results were obtained with the ground powder, and with the Cy5.5-siRNA/chitosan complex, although small but statistically significant differences weredetected in the Cy5.5-siRNA/chitosan complex. Therefore, the supercritical CO2 technique andmanual grinding did notcause a marked change in the physicochemical properties of the siRNA/chitosan complex.

Stability of siRNA in Lung From electrophoresis, the band corresponding to the siRNA was shifted from the po-sition 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 derivedfrom the lung homogenate.

Biodistribution of siRNA Following Pulmonary De-livery of the siRNA/Chitosan Powder To investigate the biodistribution of siRNA following pulmonary administra-tion, Cy5.5-siRNA was applied and the fluorescence derivedfrom 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 derivedfrom 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 alsodetected in the intestine at 6 h, which might show the parts

Fig. 1. Scanning Electron Micrographs of the siRNA/Chitosan Powders Prepared by the Supercritical CO2Technique

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

Fig. 2. Integrity of siRNA in the siRNA/Chitosan Powders Prepared by the Supercritical CO2Technique

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).Statisticallysignificantdif-ferences compared with before (** p<0.01) and after († p<0.05).

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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 pow-der. These results strongly indicated that chitosan prolonged the retention of siRNA in the lungs.To further examine the biodistribution, the mean fluores-

cence 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 thefluorescence intensity ofCy5.5-siRNAde-creased approximately 65% with the formation of a complex with chitosan in water (data not shown), that in the lung com-partment 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

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

Fig. 5. Biodistribution of siRNA Following Pulmonary Delivery of the siRNA/Chitosan Powder into Mice(A)Optical imageof thefluorescencederived fromCy5.5 inmice followingpulmonarydeliveryof nakedCy5.5-siGL3SL,Cy5.5-siGL3/ChSL, andCy5.5-siGL3/Ch

DP intomice.Thefluorescencederived fromCy5.5 inmicewasdetectedusing 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 3.5cmandheightof3.6cmfor thenon-lungcompartment.These sizesweredetermined fromadissectedmouse. (C)Timecourseoffluorescence intensityderived fromCy5.5inthelungcompartmentfollowingpulmonarydeliveryofnakedCy5.5-siGL3SL,Cy5.5-siGL3/ChSL,andCy5.5-siGL3/ChDPintomice.(D)Timecourseoffluo-rescence 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 intomice.(E)TimecourseoflungfluorescencelocalizationderivedfromCy5.5followingpulmonarydeliveryofnakedCy5.5-siGL3SL,Cy5.5-siGL3/ChSL,andCy5.5-siGL3/Ch DP into mice. Each value represents the mean±S.D. (n=3).StatisticallysignificantdifferencescomparedwithnakedCy5.5-siGL3SL(**p<0.01; * p<0.05), and with Cy5.5-siGL3/Ch SL (‡ p<0.01; † p<0.05).

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complex formed on the lung epithelial surface after the dis-solution of the powder in the lungs. Moreover, it might partly explain why the fluorescence intensity in the non-lung com-partment was higher for 0.5 h following the pulmonary deliv-ery of the Cy5.5-siRNA/chitosan powder (Fig. 5D). From 1 h after the pulmonary administration, on the other hand, both the solutionandpowder tended toexhibit lessfluorescence inthe 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 amongmice treated with each formulation. This was considered due to difficultywith the pulmonary delivery of each formulationat an exact dose because of the influences of spontaneousbreathingandmucociliaryclearancebackflow.Tocompensate,therefore, lung fluorescence localization was calculated fromthefluorescence intensity in lungandnon-lungcompartmentsin each mouse (Fig. 5E). From this evaluation, the prolonged retention of Cy5.5-siRNA in the Cy5.5-siRNA/chitosan solu-tionandCy5.5-siRNA/chitosanpowderwasclearlyconfirmedfrom 1 h following the pulmonary administration.

In-Vivo Gene Silencing by the siRNA/Chitosan Pow-der For the in-vivo gene silencing experiment, mice bearing colon26/Luc cells were subjected to imaging in vivo. The lu-minescence in the lungs increased markedly with time, corre-sponding to tumor growth (Fig. 6). On days 2 and 3 after the first treatment, the siGL3 (siRNAspecifically recognizing thefirefly luciferase gene)/chitosan powder significantly inhibitedthe increase in luminescence in the lungs compared with no treatment,while thesiRL(notspecific to thefirefly luciferasegene)/chitosan powder and siGL3/chitosan solution did not. These results strongly indicated that the siRNA/chitosan pow-

der exhibited the effective and specificgene silencing againstthe tumor cells metastasized in the lungs of mice.

DISCUSSION

In this study, we used a supercritical CO2 technique toprepare an inhalable dry siRNA/chitosan powder, and dem-onstratedapowerfulandspecific in-vivo gene silencing effect following pulmonary administration into mice. To our knowl-edge,thisisthefirstreportabout in-vivo gene silencing by an inhalable dry siRNA powder.

Preparing a powder without a loss of siRNA integrity is es-sential for effective pulmonary gene silencing by siRNA DPI. Preliminary experiments revealed that no electrophoretic band corresponding to siRNA could be detected in the siRNA pow-der prepared without chitosan (data not shown), suggesting that the supercritical CO2 techniquecausedthedestabilizationof siRNA. Tservistas et al. clarified that the acidic conditionscaused by supercritical CO2 with water destabilized pDNA during production of the powder.25) Thus, similar destabiliza-tion 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 pKa 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 pro-duced by the supercritical CO2technique.

Besides the loss of siRNA integrity, the change in the physicochemical characteristics of the complex is also an im-portantconcernformaintainingtransfectionefficiency.Inthis

Fig. 6. In-Vivo Gene Silencing in Mice by the siRNA/Chitosan Powder(A)Opticalimageoflungluminescencecorrespondingtofireflyluciferaseactivityinmicebearingcolon26/Luccellsfor3dafterthefirsttreatmentwithsiGL3/ChSL,

siGL3/ChDP, and siRL/ChDP. Colon26/Luc cells were intravenously injected into the tail. The luminescence corresponding to firefly luciferase activity was detectedusing 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 tofirefly luciferase activity inmice bearing colon26/Luc cells for 3d after thefirst treatmentwith siGL3/ChSL, siGL3/Ch DP, and siRL/Ch DP. Each value represents the mean±S.D. (n=3–5).StatisticallysignificantdifferencescomparedwithN.T.(**p<0.01; * p<0.05). N.T.; no treatment.

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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 gener-ate conformational flexibility. For detecting the electropho-retic 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 corre-sponding 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 ourhypothesis in part.

For achieving effective gene silencing by siRNA in vivo, it is important to understand the biodistribution and pharmaco-kinetics of siRNA. It has been clarified that naked siRNA isunstable in blood because of degradation by endonucleases and that when injected intravenously, siRNA is mostly ac-cumulated in the kidneys and excreted in urine.4–6) However, there are few reports about the biodistribution and pharma-cokinetics of siRNA following pulmonary administration. In this study, we investigated the fluorescence derived fromCy5.5-siRNA in vivo. Following the pulmonary administration ofnakedCy5.5-siRNAintomice, thefluorescence transferredfrom 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 fromCy5.5was detected at the positionof Cy5.5-siRNA, suggesting that Cy5.5 could stably bind to the end of siRNA in lung (data not shown). These results indi-cate that a large part of the Cy5.5-siRNA detected in the lung compartment would have kept its integrity. On the other hand, thefluorescencedetected in thenon-lung compartmentwouldbe derived from not only Cy5.5-siRNA but also the parts de-graded by endonucleases or free Cy5.5. In a pharmacokinetic study of radiolabeled naked siRNA following pulmonary ad-ministration 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 mo-lecular 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 follow-ing 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 silenc-ing 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/chi-tosan complex. The molecular weight and degree of deacety-lation 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 exten-sive deacetylation (84%) was used, which was soluble in acid-ic 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 proper-ties 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 internaliza-tion 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 silencingefficacywithouraqueousformulation.Thiswasalsoconsistent 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 sup-porting the low transfection efficiency of the siRNA/chitosancomplexinouraqueousformulation.

Interestingly, the siRNA/chitosan powder exhibited effec-tiveandspecificgenesilencingagainst the tumorcellsmetas-tasized 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

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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 thefluorescence intensity in the lungcompart-ment 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/chitosancomplex should be rapidly taken up into the lung epithelial cells,whichwassupposedbythehigherfluorescenceintensityderived 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 par-ticles of 1–5 µm are considered suitable for delivery by inhala-tion.43) On the other hand, particles of similar geometric size generate a strong adhesion force, resulting in poor dispersibil-ity.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 techniquehad a rectangular shape,witha 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 drysiRNA/chitosanpowders,a spray-freeze-drying techniquemight 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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