Noninvasive delivery of small inhibitory RNA and other reagents to pulmonary alveoli in mice

Donald Massaro,1 Gloria DeCarlo Massaro,2 and Linda Biadasz Clerch2

Lung Biology Laboratory, Departments of 1Medicine and 2Pediatrics, Georgetown University School of Medicine, Washington, District of Columbia 20057-1481

Submitted 1 March 2004 ; accepted in final form 25 June 2004


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A technically easy, noninvasive means of delivering molecules to alveoli, which act selectively or specifically in the lung, would be experimentally and therapeutically useful. As proof of principle, we took advantage of the spreading ability of pulmonary surface active material (InfaSurf), mixed it with elastase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) small inhibitory RNA (siRNA), or all-trans retinoic acid (ATRA), and instilled microliter amounts of the mixture into the nose of lightly anesthetized mice. One instillation of elastase caused diffuse alveolar destruction (emphysema) demonstrating widespread alveolar delivery. A single nasal instillation of GAPDH siRNA, compared with scrambled GAPDH siRNA, lowered GAPDH protein in lung, heart, and kidney by ~50–70% 1 and 7 days later. To test the possibility of lung-specific delivery of a potentially therapeutic drug, we administered ATRA and monitored its effect on expression of cellular retinol binding protein (CRBP)-1 mRNA, whose translation product is a key molecule in retinoid metabolism. Given intranasally, ATRA elevated CRBP-1 mRNA 4.3-fold in a lung-specific manner. The same dose and dose schedule of ATRA given intraperitoneally increased CRBP-1 mRNA only ~1.8-fold in lung; intraperitoneally administered ATRA elevated expression of CRBP-1 mRNA 1.7-fold or more in brain cortex, cerebellum, and testes, thereby increasing the risk of untoward effects. This simple noninvasive technique allows regulation of specific proteins in the lung and lung-specific delivery of reagents of experimental and potentially therapeutic importance.

bronchopulmonary dysplasia; emphysema; brain; kidney; retinoic acid


PULMONARY SURFACTANT (surface active material or SAM), a lipoprotein with great ability to spread, lines the alveoli (3, 4, 8), prevents alveolar collapse at low lung volume, and diminishes alveolar opening pressure (1, 9). Very prematurely born babies may develop bronchopulmonary dysplasia (BPD), which is characterized, in part, by an inadequate amount of SAM (1), pulmonary inflammation, excess matrix metalloproteinase activity, and arrested alveolar development (23). Exogenous SAM, delivered into the trachea, is commonly used to augment endogenous surfactant in very prematurely born babies (23). Anti-inflammatory corticosteroids given systemically are also used in the treatment of BPD (23), but they markedly impair alveolus formation in newborn rats (2, 16) and mice (11) and, hence, might do the same in humans. All-trans retinoic acid (ATRA) prevents the impairment of alveolus formation by corticosteroids in rats (15) and therefore might do the same in prematurely born babies; however, retinoids carry their own risks, especially on the developing brain (17, 21). These considerations of potential therapy and the need to easily alter specific gene expression in the lung for experimental purposes led us to test the hypothesis that reagents could be effectively delivered to the alveolus in a technically simple, noninvasive manner, using SAM as the delivery vehicle.


    MATERIALS AND METHODS
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Animal studies. Adult male C57BL/6J mice were purchased from Jackson Laboratory. At Georgetown University, they were housed three to four per cage in the Department of Comparative Medicine on a 12:12-h light-dark schedule and were allowed food (Ralston Purina Chow 5001) and tap water ad libitum. We killed mice by cutting large vessels in the abdomen after establishing a surgical level of anesthesia with xylazine (~10 mg/kg) plus ketamine (~75 mg/kg). All procedures were approved by the Georgetown University Animal Care and Use Committee and comply with the National Institutes of Health Guidelines.

We lightly anesthetized mice with xylazine (~3 mg/kg) plus ketamine (~25 mg/ml), mixed one of three reagents with SAM (InfaSurf), and instilled SAM plus the reagents, or SAM alone, into the nasal orifices of mice using a microliter pipetter; a total volume (SAM + reagent) of 0.5–1.0 µl per gram body mass was instilled. The reagents administered were porcine pancreatic elastase (Elastin Products), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) small inhibitory RNA (siRNA) plus RNasin (Ambion), scrambled GAPDH siRNA plus RNasin, or ATRA (Sigma).

Mice given elastase or SAM alone were killed 2 wk after a single intranasal instillation, and their lungs were fixed for histological analysis (1416). The mice were anesthetized with xylazine plus ketamine and killed by cutting large vessels in the abdomen. The trachea was intubated, the diaphragm was punctured, and cold 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4, was infused into the trachea at a transpulmonary pressure of 20 cmH2O. The trachea was ligated, the lungs were removed from the thorax, and fixation was continued at 0–4°C for 2 h; the tissue was then processed for microscopic examination as previously described in detail (1416).

Mice given GAPDH siRNA (10 µg) or scrambled GAPDH siRNA were killed 1 or 7 days after a single injection. Their lungs were perfused with ice-cold PBS, excised, snap-frozen in liquid N2, and stored at –80°C. Other organs were removed and stored in the same manner.

ATRA to be given intraperitoneally was dissolved in ethanol and placed in sesame seed oil (15, 16). Mice were injected intraperitoneally daily for 3 days with ATRA (500 µg/kg body mass) or equivolume ethanol plus oil (0.5 µl/g body mass). Other mice were given the same dose intranasally on the same schedule of ATRA added to SAM or SAM alone, without alcohol or sesame seed oil. We placed 0.5 µl/g body mass in the nasal orifices. Mice were killed 6 h after the last injection or instillation of these reagents; their lungs and other organs were excised without perfusion, snap-frozen in liquid N2, and stored at –80°C.

Western blot analysis. Protein extracts for Western blot analysis were prepared as previously described (5). Proteins were separated by electrophoresis in 12% SDS-PAGE and transferred onto Hybond ECL membranes. The membranes were incubated overnight in 1.0% nonfat milk in Tris-buffered saline-Tween (TBS-T; 0.01% Tween 20, 20 mM Tris, pH 7.6, and 137 mM NaCl), followed by incubation with anti-GAPDH monoclonal antibody (Ambion) at a dilution of 1:1,000 in TBS-T containing 1.0% nonfat milk. This antibody detects an ~36-kDa protein that was visualized by incubation for 1 h at room temperature with goat anti-mouse secondary antibody (Bio-Rad) in TBS-T containing 1.0% nonfat milk. Detection was effected with an enhanced chemiluminescence (ECL) kit (Amersham). The membranes were subsequently stripped by incubation in buffer containing 0.7% 2-mercaptoethanol, 20% SDS, and 6.25% 1 M Tris, pH 7.2. To control for loading, the stripped membranes were incubated overnight in 0.5% nonfat milk in TBS-T and probed with either goat anti-actin antibody (Santa Cruz) or goat anti-CuZn superoxide dismutase antibody (10). The protein bands were visualized by ECL with rabbit anti-goat secondary antibody (Bio-Rad). The protein bands were quantitated by laser densitometry (Molecular Dynamics) using ImageQuant software; GAPDH density was expressed as relative densitometry units per actin or CuZn superoxide dismutase.

Ribonuclease protection assay. Total RNA for ribonuclease protection assay (RPA) was isolated from lung homogenates using TRIzol reagent according to the manufacturer's instructions (Molecular Research Center) and was quantitated by absorbance at 260 nm with an extinction of 0.025 (µg/ml)–1 cm–1. RPA was performed using an RPA III kit (Ambion) following the manufacturer's instructions. To detect cellular retinol binding protein (CRBP)-1 mRNA, a single-stranded antisense probe was prepared by in vitro transcription with a Riboprobe kit (Ambion) as previously described (20) using a 500-bp ClaI/XbaI fragment of rat liver CRBP-1 cDNA provided by Dr. Frank Chytil (Vanderbilt Univ. School of Medicine, Nashville, TN) and subsequently subcloned in pGEM7Z (20). As an internal standard, we used pt7 RNA 18s antisense control template (Ambion), which contains 80 bp of a highly conserved region of ribosomal RNA gene. Radiolabeled 18s RNA was prepared by in vitro transcription using the Megashortscript kit (Ambion). RNA was hybridized with antisense CRBP-1 RNA and 18s RNA probes, and the intensity of the protected bands was measured, as previously described (20).

Statistical analysis. The means ± SE of each group of measurements was calculated, and an unpaired, two-tailed t-test analysis was performed to test the statistical significance of the difference between means of two groups (StatMost Statistical Analysis and Graphics version 2.58, DataMost).


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One intranasal instillation of elastase in SAM compared with equivolume SAM alone caused, depending on the dose, mild or severe diffuse emphysema (Fig. 1). This simple means of inducing emphysema could be easily used in newborn animals to explore the comparative susceptibility of newborn and adult animals to the development of emphysema and to determine the effect of neonatal alveolar destruction on lung function and architecture in later life. Furthermore, this easy noninvasive way of delivering elastase allows repeated instillations over time to mimic repeated elastase loads associated with recurrent pulmonary infections in people with chronic obstructive pulmonary disease (19).



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Fig. 1. Dose-dependent diffuse pulmonary emphysema produced in mice by intranasal administration of elastase in pulmonary surfactant. Mice were very lightly anesthetized with xylazine-ketamine. Pulmonary surfactant (1.0 µl/g body mass) alone (A), or equivolume elastase plus surfactant, at a dose of 3.0 µg/g body mass (B), or 4.5 µg/g body mass (C) was placed in the nostrils of mice. Two weeks later, mice were killed and their lungs were fixed at a transpulmonary pressure of 20 cmH2O. Bar scale = 50 µm.

 
One intranasal instillation of GAPDH siRNA in SAM lowered the lung concentration of GAPDH protein 50% at 24 h and 67% at 7 days (not tested later; Table 1). GAPDH protein in heart was diminished 40% at 24 h by instillation of GAPDH siRNA, but the intergroup values were not statistically significant at 7 days (Table 1). The concentration of GAPDH protein in kidney was diminished 40% at 24 h and 60% at 7 days (Table 1). Instillation of GAPDH siRNA did not alter the concentration of GAPDH protein in liver, cerebral cortex, cerebellum, or testes (Table 1).


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Table 1. GAPDH siRNA administered intranasally in pulmonary surfactant decreases GAPDH protein

 
We chose to use GAPDH siRNA in these proof-of-principle experiments because it is a commercially available siRNA of proven effect (Ambion). Our results with siRNA indicate naked siRNA can be delivered to the alveolus and that it can substantially depress the concentration of the target protein (Table 1, Fig. 2). Furthermore, as in the original studies in Caenorhabditis elegans (7), the effect of the siRNA extended beyond the organ at which it was targeted, i.e., beyond lung of mice and intestine of C. elegans. It is thought that a protein, SID-1, is essential and responsible for systemic spread in C. elegans (6). During the preparation of this manuscript, a report by Zhang et al. (22) demonstrated heme oxygenase-1 siRNA delivered intranasally without SAM caused lung-specific depression of heme oxygenase-1 for at least 72 h. Their work shows SAM is not essential for delivery to the alveolus of material placed in the nose. However, we have found aqueous material without SAM takes much longer to leave the nose than SAM, thereby increasing the extent of nasal absorption and potential nasal damage, e.g., from elastase.



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Fig. 2. Western blot analysis of scrambled small inhibitory RNA (siRNA) and siRNA. Mice were very lightly anesthetized with xylazine-ketamine. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) siRNA or scrambled siRNA were mixed with surface active material (SAM) and instilled into the nasal orifices of the mice. Mice were killed 24 h or 7 days later, several organs were removed, and their proteins were subjected to Western blot analysis. Each lane represents organs from 1 mouse. The brackets and lane numbers (top) designate the treatment. CuZnSOD, CuZn superoxide dismutase.

 
In considering the usefulness of this technique compared with conditional gene deletions, we acknowledge we did not achieve the equivalent of a complete knockout. However, the target protein was decreased 50–70% for a week. If that degree of downregulation of a protein does not produce a phenotype, it seems reasonable to conclude, as is concluded with classic conditional gene deletion, that there is a redundant protein, or the protein is not important to the phenotype being examined. Furthermore, there does not seem to be a reason why the expression of multiple genes could not be diminished at the same time. We think the ease of intransal delivery of reagents to the alveolus will be especially useful to evaluate the effect of changes of expression of "candidate" genes and pathways identified by microarray gene profiling in lung (5, 12), and perhaps in heart and kidney, and this may be accomplished without a massive fluid overload (18).

The intraperitoneal instillation of ATRA, one injection daily for 3 days, increased the concentration of CRBP-1 mRNA 1.7-fold or more in lung, cerebral cortex, cerebellum, and testes (Table 2, Fig. 3). The same dosage of ATRA in SAM given intranasally increased CRBP-1 mRNA in lung 4.3-fold without altering the concentration of CRBP-1 mRNA in the other organs tested.


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Table 2. ATRA administered intraperitoneally increased expression of CRBP-1 mRNA in several organs; intranasal administration caused lung-specific increased expression

 


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Fig. 3. Ribonuclease protection assay for cellular retinol binding protein (CRBP)-1 mRNA. A: mice designated i.p. were injected intraperitoneally daily for 3 days with sesame oil or eqivolume all-trans retinoic acid (ATRA; 500 µg/kg) in sesame oil. Each lane represents organs from 1 mouse. The brackets (top of each gel) indicate the treatment. B: mice designated i.n. were given SAM (1.0 µl/g body mass) intranasally or equivolume SAM plus ATRA (500 µg/kg) daily for 3 days. All mice were killed 6 h after the last administration of reagents. Each lane represents organs from 1 mouse. The brackets (top of each gel) indicate the treatment.

 
The intranasal and intraperitoneal instillation of ATRA were compared because very prematurely born babies may have alveolar collapse and high alveolar opening pressures due to an inadequate amount of surfactant (1, 9). In these infants, SAM is now provided by instillation into an endotracheal tube attached to a ventilator, which is commonly needed to assist breathing in these infants (23). To diminish pulmonary inflammation, very prematurely born infants are also commonly treated with corticosteroids administered systemically (23). In newborn rats (2, 13, 15) and mice (11), corticosteroid hormones markedly impair alveolus formation; this is prevented (14) and reversed (11) by the intraperitoneal administration of ATRA. However, there are potentially harmful effects on nonpulmonary developing organs of systemically administered ATRA in humans (17, 21). Therefore, the addition of ATRA to SAM already being instilled into the endotracheal tube as treatment in prematurely born babies, in light of the seeming absence of a systemic effect of ATRA in mice, might make a clinical trial of ATRA feasible in very prematurely born babies. Furthermore, although not examined in this paper, compared with systemic administration, corticosteroids delivered intratracheally in SAM might be as or more effective in prematurely born babies, and with fewer side effects.


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This work was supported by National Heart, Lung, and Blood Institute Grants HL-20366, HL-37666, and HL-47413.


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D. Massaro and G. D. Massaro hold a patent for the use of retinoids to treat lung diseases.


    ACKNOWLEDGMENTS
 
We thank Dr. John A. Clements, University of California, San Francisco, for suggesting we place ATRA, which comes as a powder and is poorly soluble in water, directly into SAM. We thank Zofia Opalka, Megan Ferringer, and Emma Alexander for outstanding technical help and Dr. Martin Keszler for providing InfaSurf.


    FOOTNOTES
 

Address for reprint requests and other correspondence: D. Massaro, Lung Biology Laboratory, Georgetown Univ. School of Medicine, 3900 Reservoir Road, NW, Washington, DC 20057-1481 (E-mail: massarod{at}georgetown.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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