A miniaturized nebulization catheter for improved gene delivery to the mouse lung

BACKGROUND: The available methods for administration of gene delivery systems to the lungs of small animals via nebulization have several drawbacks. These include lack of control over the delivered dose and a negative impact on the stability of the formulation. This paper describes a new nebulization catheter device for the administration of plasmid-based gene delivery systems (polyplexes) as aerosols to the mouse lung in vivo. METHODS: The physical stability of naked pDNA and polyplexes formulated with chitosan oligomers and PEI was examined following nebulization with the catheter device. We also examined the in vitro transfection efficiency of the polyplexes recovered after nebulization. Lung distribution and gene expression after administration of the selected gene delivery systems to the mouse lung were also investigated. RESULTS: In contrast to previously described nebulization methods, the structural integrity of the unprotected naked pDNA was maintained following nebulization by the catheter device, which indicates relatively mild nebulization conditions. In addition, the nebulization procedure did not affect the physical stability of the formulated polyplexes. Small volumes of the pDNA aerosol (10-20 microl) were delivered in a highly controlled and reproducible manner. The aerosol droplet size varied with the molecular weight of the polycations. Aerosol delivery via this method resulted in improved lung distribution of pDNA polyplexes and a six-fold increase in the efficiency of gene delivery in vivo over that seen with the commonly used intratracheal instillation method. CONCLUSION: The use of the nebulization catheter device provides a promising alternative for aerosol gene delivery to the mouse lung.     

Methodological optimization of polyethylenimine (PEI)-based gene delivery to the lungs of mice via aerosol application

BACKGROUND: Polyethylenimine (PEI) has been successfully used for gene delivery to the lungs of mice via aerosol application using a whole body nebulization device. In this report we optimized the design of such an aerosol device. METHODS: Aerosol devices were constructed as either serial inhalation apparatus or as a whole body nebulization chamber connected to an aerosol spacer placed in a horizontal or vertical position. PEI-based gene vectors were nebulized using a standard jet nebulizer and luciferase gene expression of various tissues was examined. RESULTS: Using a whole body aerosol device resulted in luciferase gene expression in the lungs of mice at the same level as compared with a serial inhalation apparatus. Whereas gene expression was enhanced in the presence of 5% CO(2)-in-air, anesthesia of mice strongly decreased gene expression even when mice were intubated with an intravascular cannula. Reduction of the median mass aerodynamic diameter (MMAD) of the aerosol from 3.4 to 0.27 microm by interposition of an aerosol spacer increased gene expression significantly 3-fold. Drying of the aerosol by silica gel additionally increased gene delivery significantly 3-fold. Reporter gene expression mediated by branched PEI 25 kDa was 9- and 15-fold higher as compared with linear PEIs of 22 and 25 kDa, respectively, and was dependent on the DNA concentration. Gene expression was detectable as soon as 6 h after gene vector application and reached a maximum after 72 h but was still detectable after 14 days. The presence of Zn(2+) did not increase gene expression. CONCLUSION: We propose aerosol drying as a novel and simple method of optimizing PEI-based gene delivery to the lungs.     

Distribution dynamics of perfluorocarbon delivery to the lungs: an intact rabbit model.

Motivated by the goal of understanding how to most homogeneously fill the lungs with perfluorocarbon for liquid ventilation, we investigate the transport of liquid instilled into the lungs using an intact rabbit model. Perfluorocarbon is instilled into the trachea of the ventilated animal. Radiographic images of the perfluorocarbon distribution are obtained at a rate of 30 frames/s during the filling process. Image analysis is used to quantify the liquid distribution (center of mass, spatial standard deviation, skewness, kurtosis, and indicators of homogeneity) as time progresses. We compare the distribution dynamics in supine animals to those in upright animals for three constant infusion rates of perfluorocarbon: 15, 40, and 60 ml/min. It is found that formation of liquid plugs in large airways, which is affected by posture and infusion rate, can result in a more homogeneous liquid distribution than gravity drainage alone. The supine posture resulted in more homogeneous filling of the lungs than did upright posture, in which the lungs tend to fill in the inferior regions first. Faster instillation of perfluorocarbon results in liquid plugs forming in large airways and, consequently, more uniform distribution of perfluorocarbon than slower instillation rates in the upright animals.     

Methodology for the measurement of mucociliary function in the mouse by scintigraphy.

The objective of the study was to develop a scintigraphic method for measurement of airway mucociliary clearance in small laboratory rodents such as the mouse. Previous investigations have characterized the secretory cell types present in the mouse airway, but analysis of the mucus transport system has been limited to in vitro examination of tissue explants or invasive in vivo measures of a single airway, the trachea. Three methods were used to deposit insoluble, radioisotopic colloidal particles: oropharyngeal aspiration, intratracheal instillation, and nose-only aerosol inhalation. The initial distribution of particles within the lower respiratory tract was visualized by gamma-camera, and clearance of particles was followed intermittently over 6 h and at the conclusion, 24 h postdelivery. Subsets of mice underwent lavage for evidence of tissue inflammation, and others were restudied for reproducibility of the methods. The aspiration and instillation methods of delivery led to greater distributions of deposited activity within the lungs, i.e., approximately 60--80% of the total respiratory tract radioactivity, whereas the nose-only aerosol technique attained a distribution of 32% to the lungs. However, the aerosol technique maximized the fraction of particles that cleared the airway over a 24-h period, i.e, deposited onto airway epithelial surfaces and cleared by mucociliary function such that lung retention at 24 h averaged 57% for delivery by aerosol inhalation and > or =80% for the aspiration or intratracheal instillation techniques. Particle delivery methods did not cause lung inflammation/injury with use of inflammatory cells and chemoattractant cytokines as criteria. Scintigraphy can discern particle deposition and clearance from the lower respiratory tract in the mouse, is noninvasive and reproducible, and includes the capability for restudy and lung lavage when time course or chronic treatments are being considered.     

Development of an Aerosol Model of Cryptococcus Reveals Humidity as an Important Factor Affecting the Viability of Cryptococcus during Aerosolization

Cryptococcus is an emerging global health threat that is annually responsible for over 1,000,000 infections and one third of all AIDS patient deaths. There is an ongoing outbreak of cryptococcosis in the western United States and Canada. Cryptococcosis is a disease resulting from the inhalation of the infectious propagules from the environment. The current and most frequently used animal infection models initiate infection via liquid suspension through intranasal instillation or intravenous injection. These models do not replicate the typically dry nature of aerosol exposure and may hinder our ability to decipher the initial events that lead to clearance or the establishment of infection. We have established a standardized aerosol model of murine infection for the human fungal pathogen Cryptococcus. Aerosolized cells were generated utilizing a Collison nebulizer in a whole-body Madison Chamber at different humidity conditions. The aerosols inside the chamber were sampled using a BioSampler to determine viable aerosol concentration and spray factor (ratio of viable aerosol concentration to total inoculum concentration). We have effectively delivered yeast and yeast-spore mixtures to the lungs of mice and observed the establishment of disease. We observed that growth conditions prior to exposure and humidity within the Madison Chamber during exposure can alter Cryptococcus survival and dose retained in mice.     

Anesthesia alters the pattern of aerosol retention in hamsters

General anesthesia was used to produce nonventilated areas of the lung, and aerosol inhalation was used to locate these areas, assuming that no aerosol deposits in a nonventilated region. Male Syrian golden hamsters were anesthetized with pentobarbital sodium (90 mg/kg), which reduced respiratory frequency, tidal volume, minute volume, and O2 consumption to 61, 41, 24, and 36%, respectively, of the corresponding awake levels. Awake and anesthetized hamsters were exposed to the aerosol for 30 min; then the lungs were excised, dried at total lung capacity, sliced into sections, and dissected into pieces. Autoradiographs were made of slices, and the activity and weight of pieces were determined. The evenness index (EI), a measure of the uniformity of retention, was calculated for each piece. With complete uniformity of retention, all EI's would be 1.0. In awake animals, only 0.2% (by wt) of the lungs had little or no retention (EI's less than 0.20). More particles deposited in the apex than in the base of the lungs. General anesthesia for extended periods of time with no deep breaths alters ventilation and therefore the distribution of aerosol retention. Many regions of the lungs in the anesthetized animals received few or no particles (11.6% of lungs had EI less than 0.20); however, no consistent pattern was observed in the location of these areas from animal to animal. The apex-to-base gradient for retention in these animals was also reversed. Radioactive aerosols can be used as probes to indicate the extent and distribution of nonventilated areas in the lungs.     

Effects of pleural pressure and abdominal pressure on diaphragmatic blood flow

Alveolar transfer of prostaglandin E2 (PGE2) was characterized in isolated perfused guinea pig lungs (n = 19) by measuring radioactivity appearing in the venous effluent during 30 min after intratracheal instillation of [3H]PGE2, [14C]-mannitol, and [125I]iodoantipyrine. Recovery of lipid-soluble [125I]iodoantipyrine [91 +/- 3% (SE)] after 30 min was used to estimate total 3H and 14C delivered to the exchanging region of lung at time 0. In seven control lungs, 58 +/- 4% of [14C]mannitol and 16 +/- 4% of [3H]PGE2 was retained 10 min after instillation. Neither perfusion with diphloretin phosphate (10 micrograms/ml; n = 4) nor hypothermia (5 degrees C; n = 5) significantly affected the amount of [14C]mannitol retained; however, [3H]PGE2 remaining in these lungs increased significantly to 36 +/- 4 and 53 +/- 2%, respectively. Addition of unlabeled PGE2 (200 micrograms) to the instilled solution (n = 3) increased retention of both [14C]mannitol (80 +/- 3%) and [3H]PGE2 (65 +/- 4%). Alveolar transfer of [3H]PGE2 was calculated as the difference in percent retention of [14C]mannitol and [3H]PGE2 and normalized to that of [14C]mannitol. After 10 min, alveolar transfer of [3H]PGE2 was 71 +/- 8% in control lungs but was decreased to 26 +/- 7, 10 +/- 5, and 19 +/- 6% by diphloretin phosphate, hypothermia, or unlabeled PGE2, respectively. These data suggest that alveolar clearance of PGE2 involves a saturable drug- and temperature-sensitive process.     

Effect of papain-induced emphysema on permeability of rat lung to drugs.

To investigate the effect of a papain-induced emphysema-like condition on pulmonary absorption of drugs, rats were exposed to either papain aerosol or distilled water aerosol (control) intermittently for 2 wk, and rates of drug absorption from damaged and control lungs were compared. To measure absorption rates, 0.1 ml of drug solution (0.1-10 mM) was administered through a tracheal cannula to anesthetized animals, and after various times lungs were assayed for unabsorbed compound. In absorption experiments with the lipoid-insoluble compounds, mannitol, p-aminohippuric acid, and procaine amide ethobromide, all three drugs were absorbed from the lungs about twice as rapidly in papain-treated rats as in control. In contrast, procaine amide, a relatively lipoid-soluble drug, was absorbed at the same rate in both control and papain-treated animals. The results suggest that papain-induced lung damage increases the porosity of the pulmonary epithelium.     

Airway responsiveness to inhaled and intravenous carbachol in sheep: effect of airway mucus

Excessive airway mucus can alter both the mass and site of aerosol deposition, which, in turn, may affect airway responsiveness to inhaled materials. In six prone sheep, we therefore measured pulmonary airflow resistance (RL) and cumulative aerosol deposition during five standard breaths (AD5) at base line and 3 min after inhalation challenge with 2% carbachol in buffered saline (10 breaths, tidal volume = 500 ml) or after an intravenous loading dose of carbachol (3 micrograms/kg) followed by a constant infusion of 0.3 micrograms.kg-1.min-1 with and without instillation of 20 ml of a mucus simulant (MS) into the distal end of each of the main bronchi or 30 ml of MS into the right main bronchus only by means of a flexible fiber-optic bronchoscope. Before carbachol challenge, RL did not change with MS into either both lungs or one lung only. AD5 increased from 36 +/- 2% (SE) before to 42 +/- 2% after MS instillation into both lungs (P less than 0.05) but remained unchanged after MS into one lung. After carbachol inhalation, RL increased significantly by 154 +/- 20 before and 126 +/- 25% after MS into both lungs and 162 +/- 24 before and 178 +/- 31% after MS into one lung (P less than 0.05). When the percent increase in RL was normalized for total aerosol deposition (% delta RL/AD5), the normalized values were lower after MS (3.0 +/- 0.5) than before MS (4.4 +/- 0.3) into both lungs (P less than 0.05) but were not significantly different before and after MS into the right lung only.(ABSTRACT TRUNCATED AT 250 WORDS)     

Noninvasive Intratracheal Intubation to Study the Pathology and Physiology of Mouse Lung

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular disease and for developing therapeutic intervention. With the increasing availability of knockout and transgenic derivatives, together with a vast amount of genetic information, mice provide one of the best models to study the molecular mechanisms underlying the pathology and physiology of lung diseases. Inhalation, intranasal instillation, intratracheal instillation, and intratracheal intubation are the most widely used techniques by a number of investigators to administer materials of interest to mouse lungs. There are pros and cons for each technique depending on the goals of a study. Here a noninvasive intratracheal intubation method that can directly deliver exogenous materials to mouse lungs is presented. This technique was applied to administer bleomycin to mouse lungs as a model to study pulmonary fibrosis.     

Pulmonary defense mechanisms in Boa constrictor

We studied aerosol deposition and the response to inhaled particles and irritants in lungs of Boa constrictor. Snakes which breathed submicrometric particles radiolabeled with 99mTc retained 41.4 +/- 9.9% of the aerosol in the trachea, 42.5 +/- 8.8% in the anterior faveolar regions, and 8.7 +/- 4.1% in the posterior saccular regions of the lungs. Low activity recovered in the gastrointestinal tract over a 5-h period following aerosol exposure indicated slow clearance of inhaled particles. In contrast to mammalian lungs, there are no macrophages resident on the surface of boa lungs, and uningested particles persist for up to 4 days without being phagocytized. Particles and irritant stimuli (Fe2O3, endotoxin, and N-formylmethionylphenylalanine) elicited only eosinophilic granulocytes that were not phagocytic. The numbers of these cells peaked at 24 h following exposure and declined gradually over the next 7 days. Lavage fluid from stimulated snake lungs contained many large lamellar figures continuous with tubular myelin, a form of surfactant. Very little of this material was recovered from control lungs. Response to inhaled particles and lung injury in boas increased surfactant release, elicited eosinophilic granulocytes, but did not recruit phagocytic mononuclear cells.     

Comparison of Streptococcus zooepidemicus and influenza virus pathogenicity in mice by three pulmonary exposure routes

Pulmonary infections may be induced in experimental animals by using several exposure routes. Inhalation of microbial aerosols is often viewed as the most relevant exposure route, although the comparability of either intranasal (i.n.) or intratracheal (i.t.) inoculation to aerosol inhalation is unclear. In these studies, the infection of mice with either Streptococcus zooepidemicus or influenza virus was compared following i.n., i.t., or aerosol challenge. The 50% lethal dose was determined by each exposure route for both microbes, and the organ clearance of a 50% lethal dose was determined. Mice were as or more sensitive to bacterial or viral infection following i.n. or i.t. instillation as compared with aerosol challenge. Organ clearance patterns of virus or bacteria varied slightly with exposure route.     

Comparison of Streptococcus zooepidemicus and influenza virus pathogenicity in mice by three pulmonary exposure routes.

Pulmonary infections may be induced in experimental animals by using several exposure routes. Inhalation of microbial aerosols is often viewed as the most relevant exposure route, although the comparability of either intranasal (i.n.) or intratracheal (i.t.) inoculation to aerosol inhalation is unclear. In these studies, the infection of mice with either Streptococcus zooepidemicus or influenza virus was compared following i.n., i.t., or aerosol challenge. The 50% lethal dose was determined by each exposure route for both microbes, and the organ clearance of a 50% lethal dose was determined. Mice were as or more sensitive to bacterial or viral infection following i.n. or i.t. instillation as compared with aerosol challenge. Organ clearance patterns of virus or bacteria varied slightly with exposure route.     

Investigation of an Aerosol Challenge Model as Alternative to the Intracerebral Mouse Protection Test for Potency Assay of Whole Cell Pertussis Vaccines

The current potency test for whole cell pertussis vaccines, the intracerebral mouse protection test, is still the only assay which has shown a correlation with protection in children. However, it has considerable disadvantages as it uses a severe challenge procedure and the results tend to show significant intra- and inter-laboratory variation. An alternative assay based on non-lethal aerosol challenge of mice has been investigated as a replacement for the current intracerebral mouse protection test. Evaluation of this indicated that the aerosol system allowed consistent inoculation of bacteria into mice and gave good reproducibility. The protective capacity of different vaccine preparations was distinguished by this assay. Furthermore, the viable counts of Bordetella pertussis in the lungs of challenged mice were immunisation dose-dependent, which allowed the relative potency of vaccines to be calculated. Comparison of potency of five batches of vaccine from different manufacturers assayed by both the intracerebral and the aerosol challenge methods ranked the vaccines in identical order. The results suggest that this method has potential for use as a potency test for whole cell pertussis vaccine which would result in a great reduction in the number of animals used. It would also replace the lethal challenge by a non-lethal procedure and thereby avoid the use of the severe intracerebral challenge procedure.     

The amount of arachidonic acid in the triacylglycerols of perfused hamster lungs is increased by prednisolone

Following the injection of 14C-arachidonic acid (4.1 nmol) into hamster isolated lungs about 80% of the administered radioactivity was retained by the lungs. During subsequent perfusion only a small amount of radioactivity was released to the perfusion effluent. This release was not affected by pulmonary infusion of prednisolone at 20 microM or 100 microM. In control lungs 84 +/- 1% (+/- SEM) of the retained radioactivity was recovered in the phospholipid, 13 +/- 1% in the neutral lipid and 3 +/- 1 in the free fatty acid fraction. Pulmonary infusion of prednisolone increased the amount of radiolabel in the neutral lipids. This was due to the increased amount of 14C-arachidonic acid in triacylglycerols. Prednisolone had no significant effects on the amount of 14C-arachidonate in diacylglycerols or in different phospholipids. Neither was the amount of free 14C-arachidonate in the lungs changed by prednisolone. The present study indicates that the release of arachidonic acid from triacylglycerols may be inhibited by prednisolone in hamster lungs.     

Phosphoramidon potentiates the endothelin-1-induced bronchopulmonary response in guinea-pigs

We investigated the effect of the endopeptidase-inhibitor, phosphoramidon, on the bronchopulmonary response induced by endothelin-1 in vivo or in isolated perfused lungs. In vivo aerosol administration of 1 or 3 μg/ml endothelin-1 for 2 min provoked no significant bronchopulmonary response. When awake animals were pretreated by an aerosol of phosphoramidon (0.1 mM, for 15 min), the bronchopulmonary response induced by 1 and 3 μg/ml endothelin-1 was markedly enhanced. In isolated guinea-pig lungs, aerosol administration of endothelin-1 (3 μg/ml, for 2 min) evoked a low increase in pulmonary inflation pressure. Treatment of awake animals with an aerosol of phosphoramidon before lung recollection led to a significant potentiation of the endothelin-1-induced increase in pulmonary inflation pressure. These results demonstrate that phosphoramidon potentiates the in vivo and in vitro bronchopulmonary response evoked by low doses of endothelin-1 and suggest that endopeptidase-like enzymes present in the airway tissue modulate the effect of the peptide.     

Behavior of magnetic particles in hamster lungs: estimates of clearance and cytoplasmic motility

Ferrimagnetic particles suspended in saline were instilled intratracheally into the lungs of Syrian golden hamsters. The particles were magnetized and aligned by applying an external magnetic field. Upon removal of the external field, the particles produced a remanent magnetic field from the lungs which decayed due to random misalignment of the particles (relaxation). Magnetization and relaxation measurements were performed immediately after instillation, then repeatedly during the first 24 h, and finally at intervals of several days up to 30 days after the instillation. The size of the initial remanent magnetic field immediately following each external magnetization is a measure of the amount of iron oxide in the lungs. It decreased with time, reflecting particle clearance. The rate of relaxation increased steeply during the first 12 h after the instillation and decreased slowly between the 5th and 30th day. Changes in the location of particles from extracellular to intracellular sites and movements from ectoplasmic to endoplasmic sites within cells may be responsible for the observed changes in relaxation rates with time.     

Transoral tracheal intubation of rodents using a fiberoptic laryngoscope

A fiberoptic laryngoscope which allows direct visualization of the deep pharynx and epiglottis has been developed for transoral tracheal intubation of small laboratory mammals. The device has been employed in the intubation and instillation of a variety of substances into the lungs of rats, and with minor modification, has had similar application in mice, hamsters, and guinea pigs. The simplicity and ease of handling of the laryngoscope permits one person to intubate large numbers of enflurane anesthetized animals either on an open counter top or in a glove-box, as may be required for administration of carcinogenic materials. Instillation of 7Be-labeled carbon particles into the lungs of mice, hamsters, rats, and guinea pigs resulted in reasonably consistent interlobal distribution of particles for each test animal species with minimal tracheal deposition. However, actual lung tissue doses of carbon exhibited some species dependence.     

Macromolecular synthesis in organ cultures of neonatal rat lung

Organ cultures of newborn rat lungs synthesize and accumulate DNA, RNA, collagen and noncollagenous proteins almost at a linear rate for at least 5 days. During this period the synthesis of collagen consistently exceeds the synthesis of noncollagenous proteins in a pattern similar to neonatal lung growth in vivo. Although some morphological characteristics of lung architecture are distorted after culture, fundamental structural similarities to lungs growing in intact animals are retained. When these cultures are maintained in atmospheres rich in oxygen, increased collagen synthesis is observed, a response similar to that of lungs in intact animals exposed to high oxygen concentrations in vivo. Our studies suggest that lung organ cultures may be a suitable system for investigating the biochemical aspects of lung tissue-environmental interaction.     

Application of respiratory heat exchange for the measurement of lung water.

A noninvasive method for measuring pulmonary blood flow and lung mass (called airway thermal volume), based on the measurements of lung heat exchange with environment, is described. The lungs function as a steady-state heat exchange system, having an inner heat source (pulmonary blood flow) and an external heat sink (ventilation). Sudden changes in the steady-state condition, such as caused by hyperventilation of dry air, lead to a new steady state after a few minutes. The expired air temperature difference between the initial and final steady states is proportional to pulmonary blood flow, whereas the rate at which the new steady state is achieved is proportional to airway thermal volume. The method was tested in 20 isolated dogs lungs, 9 perfused goat lungs, and 27 anesthetized sheep. The expired air temperature fall during hyperventilation was inversely proportional to the perfusion rate of the isolated lungs, and half-time of the temperature fall was proportional to the lung tissue mass. Experiments in anesthetized sheep showed that the measured airway thermal volume is close to the total mass of the excised lungs, including its residual blood (r = 0.98). Pulmonary edema and fluid instillation into the bronchial tree increased in the measured lung mass.