Factors controlling particle size during nebulization of DNA-polycation complexes.

Pulmonary gene therapy has the potential to treat or cure respiratory diseases such as cystic fibrosis. Much work has focused on the delivery of genes to the lung using viral vectors with varying degrees of success. Viral vectors are problematic and undesirable for use in the lung because they can provoke an acute immune response. This study has focused on the characterization of nonviral, polymer-based gene vectors for use with nebulizers. Calf thymus DNA has been used as a model, and was complexed with each of the three polycations; 22 kDa linear polyethyleneimine, 25 kDa branched polyethyleneimine, and 29.5 kDa polylysine using water, glucose solution, and phosphate-buffered saline (PBS) as carrier liquids. Fourier transform infrared spectroscopy has shown that the DNA retains the B form during the complex formation. The complexes prepared at N:P ratios of 10, have been nebulized using a vibrating plate nebulizer and the particle size and Zeta potentials measured before and after nebulization. The particle size distributions of the DNA complexes prepared in water and glucose solution were unimodal before and after nebulization with a small increase in particle size following nebulization. Choice of complexing polymer is shown to have only a small effect on particle size with the dominant effect coming from the ionic character of the dispersion fluid. Complexes prepared in PBS, although originally unimodal, showed pronounced agglomeration on nebulization. With all polymers in water or glucose solution, the Zeta potential increases after nebulization, but with PBS as the carrier liquid the potential falls and is clearly associated with the observed agglomeration. Gel electrophoresis shows that the complexing polymers protect the DNA through the nebulization process in all cases.     

Stabilizing protein formulations during air-jet nebulization

Whilst some proteins can be effectively administered to the lungs using a nebulizer, others, such as lactate dehydrogenase (LDH) are degraded during air-jet nebulization. In order to deliver LDH by nebulization a protective delivery system or carrier may therefore be appropriate. The aim of this study was to produce and characterize a formulation of LDH, which retains enzyme activity during nebulization. Chitosan, a biocompatible, biodegradable and bioadhesive polysaccharide polymer, was included in the formulations studied as a potential protective agent. Complexes of LDH with chitosan of different molecular weights and concentrations were assessed for size, zeta potential, aerosol droplet size and delivery from a jet nebulizer. The highest molecular weight chitosan had the greatest complex size and a net positive charge of +29.7mV. Jet nebulization resulted in aerosol droplets with median size in the range 2.36-3.52μm. Nebulization of LDH solution resulted in enzyme denaturation and reduced activity. The stability of LDH was greatly improved in formulations with chitosan; with greater than 50% total LDH available in a nebulizer delivered to the lower stage of a two-stage impinger, with up to 62% retained enzyme activity. The nonionic surfactant Tween 80 also improved the stability of LDH to nebulization and had an additive protective effect when included, with chitosan, in formulations. These findings suggest chitosan may be a useful excipient in the preparation of stable protein formulations for jet nebulization.     

Comparison of lung deposition of amikacin by intrapulmonary percussive ventilation and jet nebulization by urinary monitoring.

The intrapulmonary percussive ventilation (IPV), frequently coupled with a nebulizer, is increasingly used as a physiotherapy technique; however, its physiologic and clinical values have been poorly studied. The aim of this study was to compare lung deposition of amikacin by the nebulizer of the IPV device (Percussionaire; Percussionaire Corporation; Sandpoint, ID) and that of standard jet nebulization (SST; SideStream; Medic-Aid; West Sussex, UK). Amikacin was nebulized with both devices in a group of five healthy subjects during spontaneous breathing. The deposition of amikacin was measured by urinary monitoring. Drug output of both devices was measured. Respiratory frequency (RF) was significantly lower when comparing the IPV device with SST (8.2 +/- 1.6 breaths/min vs. 12.6 +/- 2.5 breaths/min, p < 0.05). The total daily amount of amikacin excreted in the urine was significantly lower with IPV than with SST (0.8% initial dose vs. 5.6% initial dose, p < 0.001). Elimination halflife was identical with both devices. Drug output was lower with IPV than with SST. The amount of amikacin delivered to the lung is sixfold lower with IPV than with SST, although a lower respiratory frequency was adopted by the subjects with the IPV. Therefore, the IPV seems unfavorable for the nebulization of antibiotics.     

Accounting for radioactivity before and after nebulization of tobramycin to insure accuracy of quantification of lung deposition.

The ability to predict drug deposition of inhaled drugs used in cystic fibrosis (CF) is important if there is a need to target specific doses of drug to the lungs of individual patients. The gold standard of measuring pulmonary deposition is the quantification of an aerosolized radiolabel either mixed with the drug solution or tagged directly to the compound of interest. Accuracy of the quantification could be assured if there is agreement between the amount of radioactivity before and after administration. Before administration, the radiolabel is concentrated in the well of the nebulizer, whereas after administration, it is distributed throughout the nebulizer, the expiratory filter and connectors, and the upper airway, stomach, trachea, and lung. Not only is the geometry of the distribution that is presented to the gamma camera different, but there are different attenuation factors for the various body tissues. The primary aim of this study was to evaluate the accuracy of the quantification of deposition. Secondary goals were to compare in vitro nebulizer performance with that measured in vivo during the deposition study. Eighty milligrams of tobramycin and technetium bound to human serum albumin was administered to 10 normal adults using a Pari LC Jet Plus (Pari Respiratory Equipment, Inc., Richmond, VA) breath-enhanced nebulizer. Techniques were developed that allowed for the accounting of 99 +/- 2% of the initial radioactivity. The fraction of the rate of lung deposition to total body deposition was the in vivo respirable fraction (0.62 +/- 0.07), which closely agreed with in vitro measurements of respirable fraction (0.62 +/- 0.04). Drug output measured from the change in weight and concentration in the nebulizer systematically overestimated drug output measured by the deposition study. The results indicate that 11.8 of the initial 80 mg would be deposited in the lungs. This technique could be adapted to accurately quantify the amount of deposition on any inhaled therapeutic agent, but caution must be used when extrapolating performance of a nebulizer on the bench to expected deposition in patients.     

A history of nebulization.

The simplest and most natural route of drug delivery to the lungs is the inhaled one. From the historical and medical point of view, it was a Greek, Pedanus Discorides, the father of the science of pharmacy, who, during the first century prescribed inhaled fumigation. Pipes were also used to inhale hallucinogenic substances. All shamans knew the psychotropic effects of poisonous plants such as Datura stramonium, especially Red Indians, in their peace calumets; but Indians of Madras used fumigations of Datura ferox to treat asthma. Since 1803, this therapeutic was imported in Great Britain and cigarettes with leaves of datura were used by asthmatics until 1992. In the middle of the nineteenth century, to treat grapevines diseases and in response to the fashion of inhaling thermal waters, spray technology was developed for the effervescent waters at the thermal spas. The onslaught of tuberculosis, similar to AIDS a century later, brought back into practice the inefficacious use of antiseptic aerosol therapy. With the discovery of adrenaline, ephedrine aerosols enjoyed a rebirth. The perfecting of jet nebulizers by R. Tiffeneau, father of FEV1 and M.B. Wright, father of peak-flow, allowed a better practice of inhalotherapy. In 1949, the United States, ultrasonic nebulizers made their first appearance in the form of humidifiers, but doctors were quick to add medications to produce therapeutic aerosols. After 150 years, with the improvement of nebulizer systems and new nebulized medications, the nebulization story is still not concluded.     

Lung deposition of mannitol powder aerosol in healthy subjects.

Mannitol as a dry powder aerosol is used for bronchoprovocation testing and to enhance mucus clearance in people with excessive airway secretions. The dose and distribution of the deposited aerosol in the lung was investigated using fast single photon emission tomography (SPECT) imaging. Mannitol powder (3 microm particle size) was produced by spray drying and radiolabeled with (99m)Tc-DTPA. Approximately 60 mg of radiolabeled mannitol (containing 52-68 MBq of (99m)Tc-DTPA) was administered to 10 healthy subjects using the Inhalator dry powder inhaler (DPI), and SPECT images (1 min each) were collected. Thirteen percent to 31% of the dose of mannitol loaded in the inhaler deposited in the lungs and the deposited dose correlated positively with the peak inhalation air flow. The regional aerosol lung distribution, as expressed by the penetration index (i.e., ratio of peripheral to central deposition in the lung) varied from 0.31 to 0.88, which however showed no dependency on any flow parameters. The variation in response to the same dose of mannitol within the asthmatic population may in part be explained by these findings.     

Factors affecting aerosol performance during nebulization with jet and ultrasonic nebulizers

Nebulization of aqueous solutions is a convenient delivery system to deliver drugs to the lungs because it can produce droplets small enough to reach the alveolar region. However, the droplet size might be affected by the changes in the temperature and the concentration of the nebulizing solution in the reservoir during nebulization. In this study, the changes in the droplet size over the nebulization time using a PariBoy air-jet and a Multisonic ultrasonic nebulizer have been studied. The findings were related to changes in the temperature, concentration, surface tension, viscosity and saturated vapour pressure of the nebulizing solution. By using the jet nebulizer, an increase in the droplet size followed by a decrease has been observed. This observation could be attributed to the approx. 7 degrees C reduction of the temperature during the first 2 min in the jet nebulizer reservoir which increased the viscosity of the nebulizing solution. After this initial period of time, the increasing drug concentration induced a reduction of the surface tension and, consequently, a decrease in the droplet size. However, with the ultrasonic nebulizer a temperature increase of approx. 20 degrees C during the first 6 min in the nebulizing solution was observed leading to a decrease in droplet size, viscosity and surface tension and an increasing saturated vapour pressure. This again led to smaller average droplet sizes.     

Flow defects in wet powder mass extrusion

Surface impairment, in the form of the flow defects roughness and sharkskinning, has been shown to occur with microcrystalline cellulose/lactose/water mixes on extrusion under varying process conditions. These defects result from the imbalance of stresses at the outer wall of the extrudate on leaving the die and can be avoided by changing the process conditions that are known to affect these stresses, e.g. the length of the die, the velocity of throughput and the moisture content of the formulation.     

Comparison of lung deposition of a solution after nebulization by three commonly used portable nebulizers

The efficacy of small portable nebulizers is often assumed by patients, pharmacists and physicians. Three healthy volunteers inhaled a mixture of sodium cromoglycate solution mixed with aqueous technetium-99 nebulized by each of three nebulizers and the distribution of radioactivity was detected by gamma scintigraphy. Unexpectedly large differences in the total amount of tracer delivered to the lungs were detected. The possible mechanisms and implications are discussed but it is clear that more information on the performance of individual nebulizers should be available.     

Lung deposition of droplet aerosols in monkeys

Nonhuman primates are often the animal models of choice to study the infectivity and therapy of inhaled infectious agents. Most animal models for inhaled infectious diseases use aerosol/droplets generated by an atomization technique such as a Collison nebulizer that produces particles in the size range of 1 to 3 microm in diameter. There are few data in the literature on deposition patterns in monkeys. Our study was designed to measure the deposition pattern in monkeys using droplets having diameters of 2 and 5 microm using an exposure system designed to expose monkeys to aerosols of infectious agents. Six cynomolgus monkeys were exposed to droplets. The aerosol solution was generated from a Vero cell supernate containing DMEM + 10% fetal bovine serum tagged with Tc-99m radiolabel. Collison and Retec nebulizers were used to generate small and large droplets, respectively. The particle size (as determined from a cascade impactor) showed an activity median aerodynamic diameter (AMAD) of 2.3 and 5.1 microm for the Collison and Retec nebulizer, respectively. The animals were anesthetized, placed in a plethysmography box, and exposed to the aerosol. The deposition pattern was determined using a gamma camera. Deposition in the head airways was 39% and 58% for 2.3- and 5.1-microm particle aerosols, respectively, whereas the deposition in the deep lung was 12% and 8%, respectively. This information will be useful in developing animal models for inhaled infectious agents.     

Lung surfactant phospholipids inhibit the uptake of respirable microspheres by the alveolar macrophage NR8383

Fluorescent poly(lactic-co-glycolic acid) microspheres of a respirable size were fabricated for use in a fluorescent activated cell sorting assay utilizing the continuous alveolar macrophage NR8383. This is a suitable model of alveolar phagocytosis, which permitted an investigation of the influence of phospholipid structure on the inhibition of phagocytosis of microspheres. Phospholipid inhibition was found to be independent of phosphatidylcholine alkyl chain length. Head group effects were investigated by studies employing phosphatidyl-choline, -serine, and -ethanolamine, and inhibition was shown to be independent of head group. Closer modelling of the lung environment by co-culturing NR8383 on A549 alveolar epithelium showed type II secretions to also down-regulate phagocytosis. In addition, pre-incubation with microspheres coated with dipalmitoylphosphatidylcholine reduced the uptake of a second microsphere (fluorescein isothiocyanate-labelled latex).     

In vitro deposition of the respirable fraction of dry powder inhalations determined by laser diffractometry and inertial impaction

Particle size analysis of drug and excipient is of particular interest for dry powder inhalation (DPI) formulations development and quality control. In this work, the deposition in the upper, the medium and the lower (i.e. the respirable fraction) Twin Impinger compartments of sodium cromoglycate (SCG), lactose (as excipient) and a 1:1 mixture thereof was determined by chemical analysis using a DPI device--the Micro-hale--and compared with the results obtained by laser diffractometry for the same fractions. The analytical method for the SCG determination consisted of ultraviolet spectrophotometry and, for the lactose, high performance liquid chromatography with refractive index detection was used. Laser diffractometry as a quickly operating routine method can substitute the chemical analysis in order to evaluate the respirable fraction, under the conditions of the present work and therefore making formulation development easier and quicker.     

Lung deposition in cystic fibrosis patients using an ultrasonic or a jet nebulizer.

This study was conducted to compare pulmonary deposition following inhalation with an ultrasonic and a jet nebulizer in CF patients under conditions relevant to practice. The marker substance used to estimate the relative lung bioavailability was sodium cromoglycate (SCG), which is poorly absorbed from the gastrointestinal tract, but is completely absorbed from the lungs. Ten CF patients (aged 9-21 years) used an ultrasonic nebulizer (Multisonic compact 2.4 MHz) and a jet nebulizer (Parimaster, LC Plus Turbo) in a crossover design to inhale a solution containing 20 mg of SCG and a beta(2)-agonist. Urine was collected in five fractions until 12 h p. a., and the excreted SCG was determined by means of HPLC. Prior to each inhalation, the patients' pulmonary function was measured employing a Pneumoscope. Using the ultrasonic nebulizer, the amount of SCG excreted in urine was significantly greater than that after inhalation with the jet nebulizer (1.43 +/- 0.47 mg vs. 1.04 +/- 0.47 mg; p = 0.002), despite the larger residual volume in the ultrasonic nebulizer. The absorption half-life for SCG following ultrasonic nebulization was significantly shorter when compared with jet nebulization (84 +/- 14 min vs. 101 +/- 19 min; p = 0.005), being suggestive of a more peripheral deposition. Furthermore, an inverse relationship was found between absorption half-life and FEV(1) (% pred.) (r = -0.655, p = 0.04) or MMEF(75/25) (% pred.) (r = -0.844, p = 0.031), but only with the ultrasonic nebulizer. In conclusion, the ultrasonic nebulizer tested when used for inhalation in CF patients was found to be at least equivalent to the jet nebulizer.     

Lung deposition after electronically breath-controlled inhalation and manually triggered conventional inhalation in cystic fibrosis patients.

The present work aimed to investigate whether lung deposition can be improved by using a device that optimizes the breathing pattern through electronic control. The relative lung deposition was estimated by inhalation of the marker substance, sodium cromoglycate (SCG), and measurement of urinary excretion of SCG. Thirteen cystic fibrosis (CF) patients (aged 8-20 years) received 20 mg of SCG as nebulizer solution by means of (a) conventional inhalation (Parimaster + Pari LC Star nebulizer, manual interrupter) and (b) electronically breath-controlled inhalation (AKITA + Pari LC Star nebulizer). Inhalations were trained and supervised by a physiotherapist. Patients were asked to provide answers to a questionnaire about the convenience of electronically breath-controlled inhalation. Urine was collected in five fractions until 12 h p.a., and the excreted SCG was determined by means of high-performance liquid chromatography (HPLC). Following breath-controlled inhalation, the amount of SCG excreted in urine was significantly greater than after conventional inhalation (2.22 +/- 0.61 mg vs. 1.63 +/- 0.59 mg, p < 0.001). The absorption half-life for SCG following breath-controlled inhalation was significantly shorter when compared with conventional inhalation (78 +/- 23 min vs. 107 +/- 29 min; p < 0.01), suggestive of a more peripheral deposition for the former. Ninety-two percent of the patients judged that the electronically breath-controlled inhalation was good or very good. In conclusion, inhalation with an electronically optimized breathing pattern yields a greater and more peripheral lung deposition of SCG compared with the manually triggered conventional nebulizer technique in CF patients with several years of aerosol inhalation experience.     

超声雾化吸入与氧气雾化吸入在妇科全麻术后卧床患者中的临床应用

目的 探讨超声雾化吸入与氧气雾化吸入在妇科全身麻醉(全麻)术后卧床患者中的临床应用效果.方法 87例妇科全麻术后卧床患者,根据雾化治疗方法不同分为对照组(42例)和观察组(45例).两组雾化药物相同,观察组患者应用氧气雾化方式,对照组采用超声雾化方式.比较两组患者治疗1周内排痰量以及治疗前后动脉血氧分压(PaO2)、动...     

Evidence-based treatment during the SARS-CoV-2 pandemic: Identifying the knowns and unknowns of nebulization

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus 19 (COVID-19), has resulted in a worldwide pandemic and currently represents a major public health issue. Among the considerations for patients with respiratory disease is the optimal delivery of inhaled bronchodilators to maximize patient care. Despite the lack of evidence, there is heightened concern about the potential risk for transmission of SARS-CoV-2 in the form of aerosolized respiratory droplets during the nebulized treatment of patients with COVID-19. In this commentary, the lack of evidence regarding nebulization and transmission of coronaviruses are discussed.     

Lung deposition patterns of MWCNT vary with degree of carboxylation

Functionalization of multi-walled carbon nanotubes (MWCNT) is known to affect the biological response (e.g. toxicity, inflammation) in vitro and in vivo. However, the reasons for these changes in vivo are not well described. This study examined the degree of MWCNT functionalization with regard to in vivo mouse lung distribution, particle retention, and resulting pathology. A commercially available MWCNT (source MWCNT) was functionalized (f-MWCNT) by systematically varying the degree of carboxylation on the particle’s surface. Following a pilot study using seven variants, two f-MWCNT variants were chosen and for lung pathology and particle distribution using oropharyngeal aspiration administration of MWCNT in Balb/c mice. Particle distribution in the lung was examined at 7 and 28?days post-instillation by bright-field microscopy, CytoViva hyperspectral dark-field imaging, and Stimulated Raman Scattering (SRS) microscopy. Examination of the lung tissue by bright-field microscopy showed some acute inflammation for all MWCNT that was highest with source MWCNT. Hyperspectral imaging and SRS were employed to assess the changes in particle deposition and retention. Highly functionalized MWCNT had a higher lung burden and were more disperse. They also appeared to be associated more with epithelial cells compared to the source and less functionalized MWCNT that were mostly interacting with alveolar macrophages (AM). These results showing a slightly reduced pathology despite the extended deposition have implications for the engineering of safer MWCNT and may establish a practical use as a targeted delivery system.     

Liposomes for drug delivery to the lungs by nebulization.

Preparation of drug-loaded freeze-dried (FD) liposomes, designed for delivery to lungs after rehydration/nebulization was investigated. Rifampicin (RIF) incorporating multilamelar (MLV) and dried rehydrated vesicles (DRV); composed of phosphatidylcholine (PC), dipalmitoyloglycero-PC (DPPC) or distearoyloglycero-PC (DSPC), containing or not Cholesterol (Chol), were prepared. Vesicles were characterized for encapsulation efficiency (EE%), size distribution, zeta-potential, stability during freeze drying (FD) and nebulization (nebulization efficiency (NE%) and retention of RIF after nebulization (NER%)). Mucoadhesion and toxicity in A549 cells was measured. RIF EE% was not affected by liposome type but lipid composition was important; Synthetic lipid vesicles (DPPC and DSPC) had higher EE% compared to PC. As Chol increased EE% decreased. Freeze drying (FD) had no effect on EE%, however trehalose decreased EE% possibly due to RIF displacement. NER% was highly affected by lipid composition. Results of NE% and NER% for RIF-loaded liposomes show that DSPC/Chol (2:1) is the best composition for RIF delivery in vesicular form to lungs, by nebulization. Mucoadhesion and A549 cell toxicity studies were in line with this conclusion, however if mucoadhesion is required, improvement may be needed.     

Cytotoxicity and DNA-damage in human lung epithelial cells exposed to respirable alpha-quartz.

Occupational exposure to respirable crystalline silica is associated with the development of silicosis, lung cancer and airways diseases. In order to assess cytotoxic effects and direct-oxidative DNA damage induced by short-term exposure to different doses of respirable alpha-quartz (NIST SRM1878a), we conducted a study using A549 cells. The cells were exposed to alpha-quartz at 25, 50, 100 microg/ml for 4 h and analysed by scanning electron microscope (SEM) and LDH release assay for cytotoxic effect evaluation. Cells were also exposed to 10, 25, 50, 100 microg/ml of alpha-quartz for 2 h and 4 h and analysed by Fpg comet test to evaluate direct and oxidative DNA damage. SEM observations of treated cells showed bleb development at lower doses and alterations of microvilli morphology at the highest dose. A slight LDH release was found only at 100 microg/ml. Fpg comet test showed a dose-related oxidative DNA damage in cells exposed for 2 h to quartz. Cells exposed for 4h at the same concentrations showed a dose-related direct DNA damage and the presence of oxidative DNA damage at lower doses. The bleb induction on cell surface evidenced by SEM at lower doses correlates with the presence of oxidative DNA damage at 4 h. The cell surface modifications observed by SEM at 100 microg/ml indicate that high doses of quartz induce more evident cytotoxic effects confirmed by LDH analysis and correlate with the genotoxicity showed by comet assay.