Ethanol injection method for hydrophilic and lipophilic drug-loaded liposome preparation

In this article, a hydrophobic (beclomethasone dipropionate; BDP) and a hydrophilic (cytarabine; Ara-C) drugs have been encapsulated in liposomes in order to be administered via the pulmonary route. For this aim, a liposome preparation method, which is easy to scale up, the ethanol injection method, has been selected. The effects of critical process and formulation parameters have been investigated. The drug-loaded liposomes were prepared and characterized in terms of size, zeta potential, encapsulation efficiency, release study, cell uptake, and aerodynamic behavior. Small multilamellar vesicles, with sizes ranging from about 80 to 170?nm, were successfully obtained. Results indicated a significant influence of phospholipid and cholesterol amounts on liposome size and encapsulation efficiency. The higher encapsulation efficiencies were about 100% for the hydrophobic drug (BDP) and about 16% for the hydrophilic one (Ara-C). The in vitro release study showed a prolonged release profile for BDP, in contrast with Ara-C, which was released more rapidly. The cell-uptake test revealed that fluorescent liposomes have been well internalized into the cytoplasm of SW-1573 human lung carcinoma cells, confirming the possibility to use liposomes for lung cell targeting. Nebulized Ara-C and BDP liposomes presented aerodynamic diameters compatible with deep lung deposition. In conclusion, the elaborated liposomes seem to be promising carriers for both Ara-C and BDP pulmonary delivery.     

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.     

RETRACTED ARTICLE: The Influence of Sodium Hyaluronate, <Emphasis Type="SmallCaps">l</Emphasis>-Leucine and Sodium Taurocholate on the Nebulization of Aqueous Betamethasone-17-Valerate Suspensions

The purpose of this research was to evaluate the variables that are suggested to influence the adsorption of the hydrophilic hyaluronic acid (HA) onto the surface of the hydrophobic betamethasone-17-valerate (BV) particles in order to formulate a nebulizable suspension. The adsorption of HA from aqueous solutions (0.04% to 0.16%, w/v) to a fixed BV concentration (0.04%, w/v) under different experimental conditions, was investigated. The method of preparation of HA-BV suspensions involved suspending BV particles either in the hydrated HA solution (method 1) or in water followed by addition of solid HA (method 2). Other variables like the time required for the adsorption to complete and temperature at which adsorption is carried out were studied. The nebulization of the suspensions was tested via an air jet nebulizer connected to a twin stage impinger. In order to improve the nebulization behavior of the optimized suspension, l-leucine or sodium taurocholate was incorporated in increasing concentrations (0.01–0.04%, w/v). The optimized suspension, having a nebulization efficiency of 33.75%, was achieved following the adsorption of HA (0.1%, w/v) onto BV particles adopting method 2 of preparation and extending for three days at 4 °C. Incorporation of either l-leucine or sodium taurocholate significantly decreased the aggregate size of the optimized suspension and consequently caused significant increases in the nebulization efficiency to reach 46.87% and 56.25%, respectively. An erratum to this article is available at .     

A High Through-put Procedure for Capturing Microsatellites from Complex Plant Genomes

A method is outlined for large-scale isolation and characterization of microsatellite sequences from complex plant genomes. The method presented here differs from the previously published procedures in the use of randomly sheared (nebulized) genomic DNA for adapter-ligation, rigorous removal of biotinylated oligos, and high-density colony blots for constructing enriched libraries. Using this method we have constructed cotton microsatellite enriched libraries with over 20% (high stringency screening) or 75% (by random sequencing). Thus far we have identified and sequenced over 500 cotton microsatellites using this procedure. The procedure can be used to generate enriched SSR libraries from genomic DNA in about one week. High throughput screening and automated DNA sequencing can be accomplished in less than one month.     

Nebulization as a tool for photosensitizer delivery to the respiratory tract

To this day, any photosensitizers for the photodynamic treatment of pulmonary illnesses have been administered intravenously. There is, however, an intrinsic difficulty in reaching the target cells or bacteria in the respiratory system. Nebulization could overcome distribution problems and alleviate side effects by delivering the photosensitizers directly to the lungs. In this study, we evaluated the viability of three photosensitizers (indocyanine green, the chlorine Photodithazine, and the porphyrin Photogem) was evaluated comparatively in a jet nebulizer. Quantitative analysis was performed by looking at the droplet size, extent of nebulization, output over time and stability of the solutions. All of the tested photosensitizers were found to be adequately nebulized. We also demonstrated the delivery of indocyanine green to the pulmonary tract and its activation with infrared light in a murine model using extracorporeal detection of fluorescence. This was an important step toward clinical implementation of the extracorporeally illuminated photodynamic inactivation of pneumonia, recently demonstrated in vivo by this research group.      

Effect of formulation on the stability and aerosol performance of a nebulized antibody

Most monoclonal antibodies (mAbs) are administered to patients intravenously to ensure high bioavailability as rapidly as possible. The airways, however, are an attractive delivery route for mAbs for the treatment of lung diseases, making it possible to increase their concentration in the target organ while limiting their systemic passage. Several challenges must be overcome for translation into clinical practice. For example, the drug and device must be paired for the efficient and reliable deposition of a pharmacologically active and safe mAb in the lung region of interest. Mesh nebulizers appear to be the most effective aerosol-producing devices for delivering large amounts of biopharmaceutical while limiting protein instability during nebulization. We used metrological and analytic methods to analyze the effect of both antibody concentration and surfactant addition on aerosol performance and antibody integrity. These two factors had a limited effect on aerosol performance, but affected antibody aggregation. The addition of surfactants to antibody formulations at concentrations appropriate for lung administration markedly reduced the formation of medium or large aggregates, as shown by dynamic light scattering and fluorescence microscopy. Aggregation was also dependent on the type of mesh nebulizer, highlighting the need to optimize drug and device together.     

Rifampicin-loaded liposomes for the passive targeting to alveolar macrophages: in vitro and in vivo evaluation

Mycobacterium avium complex (MAC), the most frequent cause of opportunistic nontuberculous pulmonary infection, is made up of a group of intracellular pathogens that are able to survive and multiply inside lung alveolar macrophages. As nebulized liposomes are reported to be effective to target antibacterial agents to macrophages, in this work we have prepared and characterized re-dispersible freeze-dried rifampicin (RFP)-loaded vesicles by using soy lecithin (SL) and a commercial, enriched mixture of soy phosphatidylcholine (Phospholipon 90, P90) with or without cholesterol. The obtained results showed that RFP could be loaded stably in SL vesicles only when cholesterol was not present in the film preparation, whereas with P90 vesicles, the highest stability was obtained with formulations prepared with P90/cholesterol 7:1 or 4:1 molar ratios. RFP-liposome aerosols were generated using an efficient high-output continuous-flow nebulizer, driven by a compressor. After the experiments, nebulization efficiency (NE%) and nebulization efficiency of the encapsulated drug (NEED%) were evaluated. The results of our study indicated that nebulization properties and viscosity of formulations prepared with the low-transition-temperature phospholipids, SL and P90, are affected by vesicle composition. However, all formulations showed a good stability during nebulization and they were able to retain more than 65% of the incorporated drug. The effect of liposome encapsulation on lung levels of RFP following aerosol inhalation was determined in rats. The in vitro intracellular activity of RFP-loaded liposomes against MAC residing in macrophage-like J774 cells was also evaluated. Results indicated that liposomes are able to inhibit the growth of MAC in infected macrophages and to reach the lower airways in rats.     

Effect of formulation on the stability and aerosol performance of a nebulized antibody

Most monoclonal antibodies (mAbs) are administered to patients intravenously to ensure high bioavailability as rapidly as possible. The airways, however, are an attractive delivery route for mAbs for the treatment of lung diseases, making it possible to increase their concentration in the target organ while limiting their systemic passage. Several challenges must be overcome for translation into clinical practice. For example, the drug and device must be paired for the efficient and reliable deposition of a pharmacologically active and safe mAb in the lung region of interest. Mesh nebulizers appear to be the most effective aerosol-producing devices for delivering large amounts of biopharmaceutical while limiting protein instability during nebulization. We used metrological and analytic methods to analyze the effect of both antibody concentration and surfactant addition on aerosol performance and antibody integrity. These two factors had a limited effect on aerosol performance, but affected antibody aggregation. The addition of surfactants to antibody formulations at concentrations appropriate for lung administration markedly reduced the formation of medium or large aggregates, as shown by dynamic light scattering and fluorescence microscopy. Aggregation was also dependent on the type of mesh nebulizer, highlighting the need to optimize drug and device together.     

Aerosol gene therapy

Gene therapy is a novel field of medicine that holds tremendous therapeutic potential for a variety of human diseases. Targeting of therapeutic gene delivery vectors to the lungs can be beneficial for treatment of various pulmonary diseases such as lung cancer, cystic fibrosis, pulmonary hypertension, alpha-1 antitrypsin deficiency, and asthma. Inhalation therapy using formulations delivered as aerosols targets the lungs through the pulmonary airways. The instant access and the high ratio of the drug deposited within the lungs noninvasively are the major advantages of aerosol delivery over other routes of administration. Delivery of gene formulations via aerosols is a relatively new field, which is less than a decade old. However, in this short period of time significant developments in aerosol delivery systems and vectors have resulted in major advances toward potential applications for various pulmonary diseases. This article will review these advances and the potential future applications of aerosol gene therapy technology.     

An improved enrichment procedure to develop multiple repeat classes of cotton microsatellite markers

The availability of a large number of molecular markers is a prerequisite, especially in cotton, for identifying a sufficient number of informative markers for mapping and genetic analysis. Despite the global importance of the cotton crop, few informative microsatellite markers are available, primarily because of the cost associated with their development. This report describes an improved and cost-effective strategy for developing microsatellite markers. Genomic DNA was randomly sheared with nitrogen gas to obtain unbiased representation of the genome, and the fragments containing microsatellites were captured by using biotinylated oligos and streptavidin-based recovery. Six libraries enriched for 14 microsatellite motifs were constructed and screened. Nearly 4900 simple sequence repeat (SSR)-containing sequences were identified, leading to the development of more than 1200 markers in a small amount of time.