Measurement of the Raman spectra and hygroscopicity of four pharmaceutical aerosols as they travel from pressurised metered dose inhalers (pMDI) to a model lung.

Particle inhalation is an effective and rapid delivery method for a variety of pharmaceuticals, particularly bronchodilation drugs used for treating asthma and COPD. Conditions of relative humidity and temperature inside the lungs are generally very different from the outside ambient air, with the lung typically being warmer and more humid. Changes in humidity, from inhaler to lung, can cause hygroscopic phase transitions and particle growth. Increasing particle size and mass can negatively affect particle deposition within the lung leading to inefficient treatment, while deliquescence prior to impaction is liable to accelerate drug uptake. To better understand the hygroscopic properties of four pharmaceutical aerosol particles; pharmaceutical particles from four commercially available pressurised metered dose inhalers (pMDIs) were stably captured in an optical trap, and their composition was examined online via Raman spectroscopy. Micron-sized particles of salbutamol sulfate, salmeterol xinafoate, fluticasone propionate and ciclesonide were levitated and examined over a range of relative humidity values inside a chamber designed to mimic conditions within the respiratory tract. The effect of temperature upon hygroscopicity was also investigated for salbutamol sulfate particles. Salbutamol sulfate was found to have significant hygroscopicity, salmeterol xinafoate showed some hygroscopic interactions, whilst fluticasone propionate and ciclesonide revealed no observable hygroscopicity. Thermodynamic and structural modelling is used to explain the observed experimental results.

Fluorescence lifetime imaging of optically levitated aerosol: a technique to quantitatively map the viscosity of suspended aerosol particles.

We describe a technique to measure the viscosity of stably levitated single micron-sized aerosol particles. Particle levitation allows the aerosol phase to be probed in the absence of potentially artefact-causing surfaces. To achieve this feat, we combined two laser based techniques: optical trapping for aerosol particle levitation, using a counter-propagating laser beam configuration, and fluorescent lifetime imaging microscopy (FLIM) of molecular rotors for the measurement of viscosity within the particle. Unlike other techniques used to measure aerosol particle viscosity, this allows for the non-destructive probing of viscosity of aerosol particles without interference from surfaces. The well-described viscosity of sucrose aerosol, under a range of relative humidity conditions, is used to validate the technique. Furthermore we investigate a pharmaceutically-relevant mixture of sodium chloride and salbutamol sulphate under humidities representative of in vivo drug inhalation. Finally, we provide a methodology for incorporating molecular rotors into already levitated particles, thereby making the FLIM/optical trapping technique applicable to real world aerosol systems, such as atmospheric aerosols and those generated by pharmaceutical inhalers.

Rapid interrogation of the physical and chemical characteristics of salbutamol sulphate aerosol from a pressurised metered-dose inhaler (pMDI).

Individual micron-sized solid particles from a Salamol® pharmaceutical inhaler are stably captured in air using an optical trap for the first time. Raman spectroscopy of the levitated particles allows online interrogation of composition and deliquescent phase change within a high humidity environment that mimics the particle's travel from inhaler to lung.

The use of absorption enhancers to enhance the dispersibility of spray-dried powders for pulmonary gene therapy.

BACKGROUND: Pulmonary gene therapy requires aerosolisation of the gene vectors to the target region of the lower respiratory tract. Pulmonary absorption enhancers have been shown to improve the penetration of pharmaceutically active ingredients in the airway. In this study, we investigate whether certain absorption enhancers may also enhance the aerosolisation properties of spray-dried powders containing non-viral gene vectors. METHODS: Spray-drying was used to prepare potentially respirable trehalose-based dry powders containing lipid-polycation-pDNA (LPD) vectors and absorption enhancers. Powder morphology and particle size were characterised using scanning electron microscopy and laser diffraction, respectively, with gel electrophoresis used to assess the structural integrity of the pDNA. The biological functionality of the powders was quantified using in vitro cell (A549) transfection. Aerosolisation from a Spinhaler dry powder inhaler into a multistage liquid impinger (MSLI) was used to assess the in vitro dispersibility and deposition of the powders. RESULTS: Spray-dried powder containing dimethyl-beta-cyclodextrin (DMC) demonstrated substantially altered particle morphology and an optimal particle size distribution for pulmonary delivery. The inclusion of DMC did not adversely affect the structural integrity of the LPD complex and the powder displayed significantly greater transfection efficiency as compared to unmodified powder. All absorption enhancers proffered enhanced powder deposition characteristics, with the DMC-modified powder facilitating high deposition in the lower stages of the MSLI. CONCLUSIONS: Incorporation of absorption enhancers into non-viral gene therapy formulations prior to spray-drying can significantly enhance the aerosolisation properties of the resultant powder and increase biological functionality at the site of deposition in an in vitro model.

The use of amino acids to enhance the aerosolisation of spray-dried powders for pulmonary gene therapy.

BACKGROUND: Pulmonary delivery of gene therapy offers the potential for the treatment of a range of lung conditions, including cystic fibrosis, asthma and lung cancer. Spray-drying may be used to prepare dry powders for inhalation; however, aerosolisation of such powders is limited, resulting in poor lung deposition and biological functionality. In this study, we examine the use of amino acids (arginine, aspartic acid, threonine, phenylalanine) to enhance the aerosolisation of spray-dried powders containing model non-viral gene vectors. METHODS: Lipid/polycation/pDNA (LPD) vectors, in the presence or absence of amino acids, were dispersed in lactose solutions, and spray-dried to produce appropriately sized dry powders. Scanning electron microscopy and laser diffraction were used to determine particle morphology and diameter, respectively. Gel electrophoresis was used to examine the influence of amino acids on the structural integrity of the LPD complex. In vitro cell (A549) transfection was used to determine the biological functionality of the dry powders, and the in vitro aerosolisation performance was assessed using a multistage liquid impinger (MSLI). RESULTS: Both gel electrophoresis and in vitro cell transfection indicated that certain amino acids (aspartic acid, threonine) can adversely affect the integrity and biological functionality of the LPD complex. All amino acids significantly increased the aerosolisation of the powder, with the arginine and phenylalanine powders showing optimal deposition in the lower stages of the MSLI. CONCLUSIONS: Amino acids can be used to enhance the aerosolisation of spray-dried powders for respiratory gene delivery, allowing the development of stable and viable formulations for pulmonary gene therapy.

Preparation of dry powder dispersions for non-viral gene delivery by freeze-drying and spray-drying.

BACKGROUND: Dry powder dispersion devices offer potential for delivering therapeutic macromolecules to the pulmonary epithelia. Previously, freeze-drying (lyophilisation) has been the accepted method for preparing dried formulations of proteins and non-viral gene vectors despite the respirability of such powders being inadequate without further processing. In this study we compare the utility of freeze-drying and spray-drying, a one-step process for producing dry and respirable powders, as methods for preparing non-viral respiratory gene delivery systems. METHODS: Lipid:polycation:pDNA (LPD) vectors comprising 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP), protamine sulphate and pEGFP-N1 in 3% lactose solution were either snap-frozen and lyophilised or spray-dried. Lyophilised powder was used as recovered or following coarse grinding. Structural integrity of dehydrated pDNA was assessed by agarose gel electrophoresis and powder particle size determined by laser diffraction. The apparent structure of the systems was visualised by scanning and transmission electron microscopy with the biological functionality quantified in vitro (A549 human lung epithelial cell line) by Green Fluorescent Protein (GFP) associated fluorescence. RESULTS: Lyophilisation produced large, irregularly shaped particles prior to (mean diameter approximately 21 microm) and following (mean diameter approximately 18 microm) coarse grinding. Spray-drying produced uniformly shaped spherical particles (mean diameter approximately 4 microm). All dehydrated formulations mediated reporter gene expression in A549 cells with the spray-dried formulation generally proving superior even when compared with freshly prepared LPD complexes. Biological functionality of the LPD dry powders was not adversely affected following 3 months storage. CONCLUSIONS: Spray-drying has utility for producing stable, efficient and potentially respirable non-viral dry powder systems for respiratory gene delivery.

An investigation of the solubility of various compounds in the hydrofluoroalkane propellants and possible model liquid propellants.

The aims of this study were to investigate descriptive parameters that may predict the solubility of compounds in the hydrofluoroalkane (HFA) propellants and to identify a model HFA propellant that is liquid at room temperature and atmospheric pressure. The solubility of 32 and 20 compounds chosen to give a wide range of physicochemical properties in HFA-134a and HFA-227, respectively, was measured. The Fedors solubility parameter and a computed log octanol water partition coefficient (CLOGP) were compared with the compounds' solubility in the HFA propellants. A total of 19 and 15 solutes had finite solubilities for HFA-134a and HFA-227, respectively, although the remaining solutes were miscible in all proportions. There was no apparent relation between solubility in HFA and the Fedors solubility parameter. This was not improved by considering the hydrogen-bonding potential of the compounds. When log solubility versus CLOGP was plotted, there was a linear relation for 16 and 12 of the compounds exhibiting a finite solubility in the HFA propellants, although four solutes (phenols) were displaced to the left of the linear relation. The remaining 3 compounds had much lower solubilities than was predicted from their CLOGPs, possibly as a consequence of their crystallinity (high melting points). Of the putative model propellants investigated (i.e., perfluorohexane (PFH), 1H-perfluorohexane [1H-PFH], and 2,2,2-trifluoroethanol), 1H-PFH was the most promising, with a linear relation between solubility in 1H-PFH and solubility in HFA propellant being observed. The solubilities in 1H-PFH were approximately 11 and 26% of those in HFA-134a and HFA-227.

Prodrug to probe solution HFA pMDI formulation and pulmonary esterase activity.

A novel salbutamol prodrug was synthesised. Solubility in HFA-134a and susceptibility to rat lung homogenate, blood and plasma esterase enzymes were investigated. Whereas salbutamol had a very low solubility in HFA-134a, the prodrug was found to be miscible in all proportions. In lung homogenate, the prodrug hydrolysed with a half-life of 45 min, re-generating approximately 17% of expected salbutamol after 8 h incubation. The use of a solution pMDI for pulmonary delivery of the salbutamol prodrug is predicted to result in liberation of salbutamol in the lungs following in vivo hydrolysis by lung esterases.