Receptor-mediated targeting of spray-dried lipid particles coformulated with immunoglobulin and loaded with a prototype vaccine.

PURPOSE: Spray-dried lipid-based microparticles (SDLM) serve as a platform for delivery of a wide variety of compounds including peptides, proteins, and vaccines to the respiratory mucosa. In the present study, we assessed the impact of IgG-mediated targeting to phagocytic cells of inactivated influenza virus formulated in SDLM, on subsequent immune responses. METHODS: SDLM were produced containing inactivated influenza virus strain A/WSN/32/H1N1 (WSN), with or without IgG. Using phagocytic antigen presenting cells (APC) and a T cell hybridoma (TcH) line specific for a dominant influenza virus epitope, we compared the in vitro responses elicited by ligand-formulated (SDLM-IgG-WSN) and non-ligand particles (SDLM-WSN). The effect of including the IgG ligand in the formulation was further characterized by measuring the immune responses of rodents vaccinated with SDLM. RESULTS: SDLM-IgG-WSN were internalized in an Fc receptor (FcR)-dependent manner by phagocytic APC that were then able to effectively present a dominant, class II-restricted epitope to specific T cells. While SDLM-WSN elicited a lower response than administration of plain inactivated virus in saline, the level of the T cell response was restored both in vitro and in vivo by incorporating the APC FcR ligand, IgG, in the SDLM. CONCLUSIONS: Incorporation of FcR ligand (IgG) in SDLM restored the limited ability of formulated virus to elicit T-cell immunity, by receptor-mediated targeting to phagocytes.

Liquid dose pulmonary instillation of gentamicin PulmoSpheres formulations: tissue distribution and pharmacokinetics in rabbits.

PURPOSE: To assess the pharmacokinetics and biodistribution of gentamicin, delivered as PulmoSpheres formulations in rabbit serum and lung tissue following intratracheal instillation in a perflubron vehicle. METHODS: Rabbits were anesthetized, intubated, and mechanically ventilated with O2 (FiO2 = 0.50). Animals were then given 5 mg/kg gentamicin either intravenously, intramuscularly (TM), or intratracheally (IT) gentamicin PulmoSpheres formulation, instilled in 1.8 ml/kg of liquid perflubron vehicle. Serum and lung lobe sections were collected at multiple time points and assayed for gentamicin content. RESULTS: Serum gentamicin levels peaked at 64.7 microg/ml, 11.2 microg/ml, and 5.0 microg/ml following intravenous, TM, and IT administration, respectively. Absolute bioavailabilitv at 8 h for IM administration was 76.8% and 57.0% when delivered IT. Although peak lung levels of drug were reached within 1 h, total lung gentamicin concentration after IT administration was more than two orders of magnitude greater than that achieved following TM administration (680,540 vs. 4,985 microg min, respectively) with significant levels of the antibiotic remaining in the lung even after 1 week. CONCLUSIONS: High levels of gentamicin in lung tissue can be achieved by instillation of a gentamicin PulmoSpheres formulation in a perflubron vehicle, termed liquid dose installation, without reaching toxic systemic levels allowing for increased local delivery of agents such as gentamicin at the site of the infection.

Novel lipid-based hollow-porous microparticles as a platform for immunoglobulin delivery to the respiratory tract.

PURPOSE: Delivery of specific antibodies or immunoglobulin constructs to the respiratory tract may be useful for prophylaxis or active treatment of local or systemic disorders. Therefore, we evaluated the utility of lipid-based hollow-porous microparticles (PulmoSpheres) as a potential delivery vehicle for immunoglobulins. METHODS: Lipid-based microparticles loaded with human immunoglobulin (hIgG) or control peptide were synthesized by spray drying and tested for: i) the kinetics of peptide/protein release, using ELISA and bioassays; ii) bioavailability subsequent to nonaqueous liquid instillation into the respiratory tract of BALB/c mice, using ELISA and Western blotting; iii) bioactivity in terms of murine immune response to xenotypic epitopes on human IgG, using ELISA and T cell assays; and iv) mechanisms responsible for the observed enhancement of immune responses, using measurement of antibodies as well as tagged probes. RESULTS: Human IgG and the control peptide were both readily released from the hollow-porous microspheres once added to an aqueous environment, although the kinetics depended on the compound. Nonaqueous liquid instillation of hIgG formulated in PulmoSpheres into the upper and lower respiratory tract of BALB/c mice resulted in systemic biodistribution. The formulated human IgG triggered enhanced local and systemic immune responses against xenotypic epitopes and was associated with receptor-mediated loading of alveolar macrophages. CONCLUSIONS: Formulation of immunoglobulins in hollow-porous microparticles is compatible with local and systemic delivery via the respiratory mucosa and may be used as means to trigger or modulate immune responses.

Hollow porous particles in metered dose inhalers.

PURPOSE: To assess the physical stability and aerosol characteristics of suspensions of hollow porous microspheres (PulmoSpheres) in HFA-134a. METHODS: Cromolyn sodium, albuterol sulfate, and formoterol fumarate microspheres were prepared by a spray-drying method. Particle size and morphology were determined via electron microscopy. Particle aggregation and suspension creaming times were assessed visually, and aerosol performance was determined via Andersen cascade impaction and dose uniformity studies. RESULTS: The hollow porous particle morphology allows the propellant to permeate freely within the particles creating a novel form of suspension termed a homodispersion, wherein the dispersed and continuous phases are identical, separated by an insoluble interfacial layer of drug and excipient. Homodispersion formation improves suspension stability by minimizing the difference in density between the particles and the medium, and by reducing attractive forces between particles. The improved physical stability leads to excellent dose uniformity. Excellent aerosolization efficiencies are also observed with PulmoSpheres formulations, with fine particle fractions of about 70%. CONCLUSIONS: The formation of hollow porous particles provides a new formulation technology for stabilizing suspensions of drugs in hydrofluoroalkane propellants with improved physical stability, content uniformity, and aerosolization efficiency.

Dissolution of multicomponent microbubbles in the bloodstream: 1. Theory.

The problem of dissolution of a bubble in the bloodstream is examined. The bubble is assumed to be filled with a mixture of a sparingly water-soluble gas (osmotic agent) and air. The dissolution of the bubble has three definite stages. In Stage 1, the bubble quickly swells in air. The swelling ratio depends on the surface tension, blood pressure, level of oxygen metabolism and initial mole fraction of osmotic agent in the bubble. In Stage 2, the osmotic agent slowly diffuses out of the bubble. The squared radius decreases nearly linearly with time, at a rate proportional to the Ostwald coefficient and diffusivity of the osmotic agent. In Stage 3, the partial pressure of the osmotic agent becomes so high that it condenses into a liquid. In order to prolong the lifetime of 5-micron bubbles in the bloodstream from < 1 s (as found with pure air), the osmotic agent must have a low Ostwald coefficient (< or = 10(-4)) and a relatively high saturated vapor pressure at body temperature (> or = 0.3 atm = 3 x 10(4) Pa).

Dissolution of multicomponent microbubbles in the bloodstream: 2. Experiment.

The effect of the nature of the filling gas on the persistence of microbubbles in the bloodstream was studied. All the microbubbles were covered with the same shells. Various perfluorocarbons and perfluoropolyethers alone and as mixtures with nitrogen were used as the filling gases. The persistence time of microbubbles in the bloodstream tau increased with the molecular weight of the filling gas, from approximately 2 min for perfluorethane, to > 40 min for perfluorodiglyme, C6F14O3, and then decreased again to 8 min for C6F14O5. An acceptable ultrasound scattering efficacy was exhibited by the filling gases with intermediate molecular weights that possessed both a high saturated vapor pressure and a comparatively low water solubility (Ostwald coefficient). On the basis of the experimental data, it is concluded that the microbubble persistence tau is controlled primarily by the dissolution of microbubbles and not by the removal of the microbubbles by the reticular endothelial system. Although the qualitative experimental trends are in good agreement with the theoretical model developed previously, there are some quantitative differences. Possible reasons for these differences are discussed.

Phospholipids as Emulsion Stabilizers

The equilibrium phase behavior and the macroemulsion type and stability of oil-water-lecithin mixtures was studied. For dioleyl phosphatidylcholine (DOPC) and n-alkanes as the oil component, the phase equilibrium is characterized by an extended inverse micellar region in equilibrium with water (Winsor II). On the other hand, n-C8F18-DOPC-water and soybean oil-DOPC-water mixtures show a three phase equilibrium of almost pure water, almost pure oil, and lamellar phase Lalpha (Winsor III). Equilibrium phases of DOPC systems can be emulsified in each other. Inverse W/O macroemulsions are favored for all the n-alkanes (C6-C14) studied; O/W emulsions are rather unstable. On the other hand, n-perfluorooctane and soybean oil produced very stable O/W emulsions. The phase behavior and emulsion stabilizing properties of an egg yolk phospholipid mixture is similar to those of DOPC. The phase equilibrium of the saturated analogue of DOPC: distearoyl phosphatidylcholine (DSPC) in mixtures with alkanes and water at room temperature is different and characterized by a Winsor III equilibrium of a Lbeta lamellar phase in a gel state, oil and water. Accordingly, the O/W emulsions are strongly favored to inverse systems. The pattern of the phase equilibrium and macroemulsion stability becomes similar to that of DOPC at elevated temperatures. The macroemulsion stability pattern versus the phospholipid packing type is discussed in relation to the recently proposed theory of emulsion stability to coalescence (Kabalnov, A. and Wennerstrom, H., Langmuir 12, 276 (1996)).

Intraocular tolerance of perfluorooctylbromide (perflubron)

PURPOSE: To determine the intraocular tolerance of perfluorooctylbromide (perflubron) in vitrectomized rabbit and pig eyes and evaluated its use as a vitreous substitute in virteoretinal surgery. METHODS: Pars plana vitrectomy was performed on 33 Dutch pigmented rabbits and 11 micro mini pigs. After vitrectomy the eyes were filled with perflubron for 2 hours, 1 week, 2 weeks, 1 month, and up to 6 months. RESULTS: No clinical, electroretinographic, or light and electron microscopic evidence of adverse effects on the retina and lens were observed. Perflubron emulsified and dispersed into small bubbles after 2-3 weeks. The lens showed mild posterior subcapsular cataracts in pig eyes after long-term retention of perflubron. CONCLUSION: These findings indicate that perflubron is safe for intraoperative and for long-term use intravitreally. However, emulsification and the breakdown into small bubbles limits the view of the retina when perflubron is used as a long-term tamponade.

Influence of perflubron emulsion particle size on blood half-life and febrile response in rats.

Perfluorochemical (PFC) emulsions are particulate in nature and, as such, can cause delayed febrile reactions when injected intravenously. This study investigated the influence of emulsion particle size on intravascular retention and on body temperature changes in unrestrained conscious rats. Concentrated (60% to 90% w/v) emulsions based on perflubron (perfluorooctyl bromide [PFOB]) with mean particle sizes ranging from 0.05 microns to 0.63 microns were tested. Rats were fitted with a chronic jugular catheter and an abdominal body temperature telemetry unit. Fully recovered, conscious rats were monitored for 24 hours after infusion (dose = 2.7 g PFC/kg). Emulsion blood half-life (T1/2) was determined from blood perflubron levels measured by gas chromatography. Emulsions with a particle size of 0.2-0.3 microns caused fevers (6 to 8 hour duration) which peaked at 1-1.5 degrees C above normal (approximately 37.5 degrees C). Fevers could be blocked by i.v. treatment with either cyclooxygenase inhibitors (ibuprofen) or corticosteroids (dexamethasone). Both intensity and duration of the temperature response, quantified by area under the temperature curve, was decreased significantly for emulsions with a particle size < or = 0.12 micron. Blood T1/2 varied inversely with particle size, and was 3 to 4 fold longer for emulsions with a mean particle size < or = 0.2 micron. Thus, smaller emulsion particles more effectively evaded the reticuloendothelial system, which resulted in longer intravascular retention, less macrophage activity, and reduced febrile responses.

Room temperature stable perfluorocarbon emulsions with acceptable half-lives in the reticuloendothelial system.

Prolonged room temperature stability (i.e. zero particle growth) can be achieved for concentrated emulsions of perflubron (perfluorooctyl bromide) or perfluorodecalin via addition of a secondary high molecular weight, lipophilic fluorocarbon component. Due to their enhanced lipophilic character, the secondary fluorocarbon components have acceptable half-lives in the organs of the reticuloendothelial system.

Analysis of Ostwald ripening in fluorocarbon emulsions by sedimentation field-flow fractionation.

The Ostwald ripening mechanism has been studied using sedimentation field-flow fractionation (SdFFF). It has been shown that significant partitioning between different sized droplets occurs for two component disperse phase emulsions, when one of the components is virtually insoluble in the continuous phase.

Fluorocarbon emulsions--the stability issue.

Long-term room temperature stability of ready-to-use concentrated fluorocarbon emulsions is necessary in order to fully exploit the therapeutic potential of fluorocarbons. Consequently, considerable efforts have been directed at investigating the physical nature of such emulsions, the mechanisms which lead to their degradation and the means of counteracting these. The particles which constitute typical fluorocarbon/egg yolk phospholipid emulsions have been identified to be surfactant-coated fluorocarbon droplets and lipid vesicles. Better understanding has been gained on the formation, structure and evolution of these particles during processing and storage. This has led to optimized formulations and processing, better control of emulsion characteristics and significantly improved stability. Molecular diffusion (Ostwald ripening or transcondensation) has been shown to be the maun mechanism of degradation when particles are less than 1 micron in diameter, even for the highly concentrated (volume fraction of fluorocarbon up to 50%) second generation fluorocarbon emulsions. Significant emulsion stabilization has been accomplished by adding fluorochemicals which are both less volatile and less water soluble, and nevertheless have an organ dwell time acceptable for intravascular use. The rate of molecular diffusion can also be reduced by decreasing the fluorocarbon/water interfacial tension; this was effectively achieved with appropriate, well-defined fluorinated surfactants. A further, novel means of stabilizing fluorocarbon-in-water emulsions makes use of mixed fluorocarbon-hydrocarbon amphiphiles which act as molecular dowels to reinforce the adhesion between the fluorocarbon phase and the lipophilic zone of the surfactant film. Both long-term room temperature stability, and particle-size control over a large range of diameter, have been achieved by applying this principle. All in all it can be said that the challenge of producing injectable fluorocarbon emulsions with long-term room temperature particle size stability has been met.

Stabilization of perflubron emulsions with egg yolk phospholipid.

Egg Yolk Phospholipid(EYP) has been used extensively as the primary surfactant in parenteral fat emulsions for many years. The simplicity, functionality and physiologic tolerance of EYP has contributed greatly to its success in the intravenous emulsion arena. The mechanism of stabilization in triglyceride emulsions is well understood; however, this is not the case with perfluorocarbon emulsions. Interfacial models, as well as emulsion stability studies, have been conducted utilizing EYP of varied composition in order to derive a structure/function relationship. Our studies indicate that minor components, total unsaturation, acyl chain length and presence of charged species have significant impact on the functional properties of EYP and the subsequent stability of the emulsion product. These findings contribute to our ability to design and manipulate natural surfactants with superior properties for use in medical applications of perfluorocarbon emulsions.

Triplet-singlet energy transfer in the complex of auramine O with horse liver alcohol dehydrogenase.

Triplet-singlet energy transfer has been studied in the complex formed between auramine O (AO) and horse liver alcohol dehydrogenase with optically detected magnetic resonance (ODMR) spectroscopy. The results show that Trp-15 and Tyr residues transfer triplet energy mainly by a trivial process, whereas Trp-314 transfers triplet energy by a Förster process with two observed lifetimes at 77 K of 170 and 50 ms. The different Förster energy-transfer lifetimes are ascribed either to quenching of the two Trp-314 residues of the dimer by a single asymmetrically bound AO or to two distinct conformations of the enzyme-dye complex with differing separations and/or orientations of donor and acceptor. Individual spin sublevel transfer rate constants are reported for the major decay component with the 170-ms Trp triplet-state lifetime; these are found to be highly selective with kxtr much greater than kytr and kztr.