Insulin nanoparticles: stability and aerosolization from pressurized metered dose inhalers.

Nanoparticles containing insulin have been produced by emulsification processes followed by freeze-drying. Purified nanoparticles were suspended in hydrofluoroalkane (HFA) 134a, using essential oils (cineole and citral) as suspension stabilizers to form pressurized metered dose inhaler (pMDI) formulations. The retention of insulin integrity after formulation processing was determined using high performance liquid chromatography (HPLC), size exclusion chromatography (SEC), circular dichroism (CD) and fluorescence spectroscopy. The results indicated that the native structure of insulin was retained after formulation processing. Aerosolization properties of the manufactured pMDI formulations were determined using a multi-stage liquid impinger. The results showed that the nanoparticles were suitable for peripheral lung deposition, with a fine particle fraction (FPF(<1.7 microm)) of approximately 45% (w/w). In conclusion, the pMDI formulations with nanoparticles containing insulin developed in this study have the potential to deliver protein therapeutics via inhalation for systemic action.

The processing of nanoparticles containing protein for suspension in hydrofluoroalkane propellants.

Nanoparticles delivered from pressurized metered dose inhalers (pMDIs) potentially offer a means of efficiently delivering proteins to the lung. Nanoparticles containing the model protein lysozyme have been produced using microemulsion and nanoprecipitation methods. Freeze-drying water in oil emulsions, with chloroform as the organic solvent, followed by washing of excess surfactant (lecithin) led to the production of lactose nanoparticles having approximately 300 nm mean size. Substitution of lactose with lysozyme led to a significant increase in the mean size of nanoparticles (645-750 nm). This may have been due to the surface activity of lysozyme which altered the emulsification properties. The retained biological activity of lysozyme increased with increased lactose concentration in the formulation, and approximately 99% biological activity was retained when 20% (w/w) lactose was used. Ethanol used in the formulation in place of chloroform changed the production process from emulsification to nanoprecipitation. A monodisperse system (mean size approximately 275 nm, polydispersity index approximately 0.1) of spherical nanoparticles containing 80% (w/w) bioactive lysozyme (retained activity 99%) was generated. The nanoparticles washed with ethanol containing DPPC, oleic acid or Span 85 (2%, w/v) could readily be dispersed in HFA 134a without further processing, and a stable suspension was formed. Lysozyme remained stable (retained biological activity 98%) even after the nanoparticles were suspended in HFA 134a. This indicates the potential of nanoparticles for delivery of proteins from HFA-based pressurized metered dose inhaler formulations.

An investigation into the influence of drug lipophilicity on the in vivo absorption profiles from subcutaneous microspheres and in situ forming depots.

Drug lipophilicity is known to have a major influence on in vivo drug absorption from intramuscularly and subcutaneously administered solutions. Indeed, chemical modification to increase drug lipophilicity is used to enable sustained drug release from solutions. In contrast to the wealth of knowledge on drug release from simple solutions, the influence of drug lipophilicity on its release from controlled release formulations, such as, microparticles and in situ forming depots, have not been systematically studied. Controlled release vehicles are designed to 'control' drug release, hence, in vitro studies show negligible influence of drug lipophilicity on release. The situation could however be different in vivo, due to interactions between the vehicle and biological tissue. We therefore investigated the influence of drug lipophilicity on its in vivo release in rats from two controlled release formulations, PLGA microparticles and in situ forming depots. Both systems exhibited a burst drug release. Subsequent to the burst release, we found that lipophilicity did not influence the rate or extent of drug absorption from the two formulations over a 10-day study period, which would imply that drug partitioning out of the depots was not the main mechanism of drug release from both formulations. This study must however be repeated with a greater number of animals to increase its power.

Combined surface pressure-interfacial shear rheology study of the effect of pH on the adsorption of proteins at the air-water interface.

The effect of pH on the adsorption of catalase and lysozyme at the air-water interface has been studied using a combined surface pressure-interfacial shear rheology technique. The results presented show that the rate of development of interfacial phenomena increases as the pH of the subphase approaches the isoelectric point of the protein under investigation. The development of the measured interfacial rheological parameters is due to an increased rate of cross-link formation within the resultant interfacial gel. The formation of the interfacial gels has been modeled using a combination of the Smoluchowski theory for the coagulation of an aerosol or fog and classic rubber elasticity theory. The enhanced rate of cross-link formation at the isoelectric point is a result of an in-surface phase separation brought about by cooperative deionization of the protein molecules near their isoelectric point. Simultaneous measurements of surface pressure and interfacial rheology have enabled us to show that the development of a gel-like interfacial network coincides with observed increases in surface pressure.

Combined surface pressure-interfacial shear rheology studies of the interaction of proteins with spread phospholipid monolayers at the air-water interface.

The adsorption of two model proteins, catalase and lysozyme, to phospholipid monolayers spread at the air-water interface has been studied using a combined surface pressure-interfacial shear rheology technique. Monolayers of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DPPG) and DPPC:DPPG (7:3) were spread on a phosphate buffer air-water interface at pH 7.4. Protein solutions were introduced to the subphase and the resultant changes in surface pressure and interfacial storage and loss moduli were recorded with time. The results show that catalase readily adsorbs to all the phospholipid monolayers investigated, inducing a transition from liquid-like to gel-like rheological behaviour in the process. The changes in surface rheology as a result of the adsorption of catalase increase in the order DPPC

Novel sustained release microspheres for pulmonary drug delivery.

A novel process for generating sustained release (SR) particles for pulmonary drug delivery is described. High purity nanoparticles of a hydrophilic, ionised drug are entrapped within hydrophobic microspheres using a spray-drying approach. Sustained release of the model drug, terbutaline sulphate (TS), from the microspheres was found to be proportional to drug loading and phospholipid content. Microspheres with a 33% drug loading exhibited sustained release of 32.7% over 180 min in phosphate buffer. Release was not significantly different in simulated lung fluids. No significant burst release was observed which suggested that nanoparticles were coated effectively during spray-drying. The absence of nanoparticles at the microsphere surface was confirmed with confocal microscopy. The sustained release microspheres were formulated as a carrier-free dry powder for inhalation, and exhibited a favourable Fine Particle Fraction (FPF) of 46.5+/-1.8% and Mass Median Aerodynamic Diameter (MMAD) of 3.93+/-0.12 microm.

Surfactant solubility and aggregate orientation in hydrofluoroalkanes.

PURPOSE: To find surfactants soluble in the two hydrofluoroalkane (HFA) propellants, HFA-134a and HFA-227ea; to compare surfactant solubility in the two propellants with those in 2H,3H-decafluoropentane (DFP) in order to assess latter's suitability as a liquid model propellant and to investigate surfactant aggregation and aggregate orientation in HFAs. METHODS: To assess surfactant solubility, HFA was added to a known amount of surfactant until dissolution was visibly apparent. An iodine solubilization method was used to determine surfactant aggregation behaviour in DFP. Fluorescence spectroscopic investigations on the surfactant orientation in aggregates were carried out in HFAs using a microviscosity sensitive fluorescent probe (1,3-dipyrenylpropane). The aim was to assess viscosity changes in the microenvironment of this lipophilic probe upon incorporation into surfactant aggregates. RESULTS: Soluble surfactants could be found among the polyoxyethylene-ethers and POE-PPO-block copolymer surfactants. Solubility in DFP appears to correlate with solubility in HFA-134a, but not HFA-227ea. Iodine solubilization indicates micellization of Brij 30 in DFP at a cmc (type II association behaviour). L-44 in DFP, on the other hand, does not exhibit a well defined cmc, but shows continuous surfactant aggregation (type I association behaviour). The fluorescence spectroscopic studies showed evidence for probe incorporation into surfactant aggregates in HFAs. CONCLUSIONS: DFP proved to be a good model for HFA-134a only. An L1-aggregate orientation was shown for surfactants in HFAs and is in marked contrast to the chlorofluorocarbon propellant where a L2-aggregate orientation exists.

Peptide washout and permeability from glyceryl monooleate buccal delivery systems.

Simultaneous evaluation of the permeation and washout of a peptide from the mucoadhesive liquid crystalline phases of glyceryl monooleate (GMO) has been investigated using a donor compartment flow-through diffusion cell. [D-Ala2, D-Leu5]enkephalin (DADLE) was incorporated into the cubic and lamellar liquid crystalline phases of GMO and applied to excised porcine buccal mucosa mounted in the donor compartment flow-through cell. Phosphate-buffered saline pH 7.4 (PBS) was pumped across the upper surface of the liquid crystalline phases to mimic salivary flow. The steady-state fluxes of DADLE and GMO from the cubic phase were significantly greater than that from the lamellar phase (P < 0.01). There was no statistical difference between the amounts of DADLE and GMO washed out from the lamellar and cubic phases (P > 0.05). The donor compartment flow-through diffusion cell was found to be a useful tool to evaluate the impact of salivary washout on mucoadhesive oral mucosal delivery systems.