Protein aggregation and enzyme activity were compared for reconstituted lysozyme particles produced by two cryogenic technologies, spray freezing into liquid (SFL) and sprayfreeze drying (SFD). The particles were characterized by enzyme activity measurements, scanning electron microscopy (SEM), light scattering, X-ray photoelectron spectroscopy (XPS) and BET specific surface area analysis. Highly porous microparticle aggregates of protein nanoparticles, observed by SEM, were produced by both processes. The smaller degree of protein aggregation and smaller losses in enzyme activity for the SFL process relative to the SFD process were due primarily to the spraying step. The higher stability of the SFL versus SFD powders was consistent with the smaller surface excess of lysozyme measured by XPS in SFL, resulting from the reduced time of exposure to the air-water interface during atomization. For pure lysozyme, the degree of aggregation and enzyme activity were comparable for lyophilization and SFL, despite the much larger particle surface area for SFL. (c) 2005 Elsevier B.V All rights reserved.
A novel high-pressure apparatus and technique were developed to measure CO2/water/solid contact angles (theta) in situ for pressures up to 204 bar. For two glass substrates with different hydrophilicities, theta increased significantly with CO2 pressure. As the pressure was increased, an increase in the cohesive energy density of CO2 caused the substrate/CO2 and water/CO2 interfacial tensions (gamma) to decrease, whereas the water/substrate gamma value increased. theta for the more hydrophobic substrate was predicted accurately from the experimental water/CO2 gamma value and an interfacial model that included only long-range forces. However, for the more hydrophilic substrate, short-range specific interactions due to capping of the silanol groups by physisorbed CO, resulted in an unusually large increase in the water/substrate gamma value, which led to a much larger increase in theta than predicted by the model. A novel type of theta hysteresis was discovered in which larger theta values were observed during depressurization than during pressurization, even down to ambient pressure. Effective receding angles were observed upon pressurization, and effective advancing angles were observed upon depressurization on the basis of movement of the three-phase contact line. The greater degree of hysteresis for the more hydrophilic silica can be attributed in part to the capping of silanol groups with M. The large effects of CO2 on the various interfacial energies play a key role in the enhanced ability of CO2 relative to many organic solvents, to dry silica surfaces as reported previously on the basis of FTIR spectroscopy (Tripp, C. P.; Combes, J. R. Langmuir 1998, 14, 7348-7352).
Solutions and emulsions containing water, hydrocarbon surfactant and CO(2) were utilized to remove post-etch residues from vias and trenches in low dielectric constant (k) patterned porous methylsilsesquioxane (pMSQ) interlayer dielectrics. The time for cleaning and rinsing was reduced to 2 min for 0.13 mu m features. The vias and trenches were cleaned with a hydrocarbon surfactant, polyoxyethylene 2,6,8-triethyl-4-nonyl ether (5b-C(12)E(8)), in water solution to provide detergency, followed by emulsification with the addition of pure CO(2). The post-etch and post-ash residues were removed from the vias and trenches both as a suspension and in a dissolved state in microemulsions and/or macroemulsions of water and carbon dioxide. The surfactant is ambidextrous in that it is interfacially active in fundamentally different types of interfaces: the solid-water, solid-M. and water-CO(2) interfaces. The residues, water and surfactant were displaced from the cleaning vessel with CO(2) assisted by gravity to reduce the rinse time from well over 10 min to approximately 1 min. The low interfacial tension produced by surfactant and supercritical CO(2) is beneficial in reducing the Laplace pressure to facilitate both penetration and removal of the cleaning solution. This benefit will become increasingly more important as the feature size decreases below 50 nm. (c) 2006 Elsevier B.V. All rights reserved.
Traditionally, finely dispersed metal catalysts have been formed by reduction of precursors within mesoporous supports. A new concept for designing catalysts with enhanced activities and selectivities is to infuse presynthesized nanocrystals with well-defined morphologies into ordered mesoporous materials. The decoupling of nanocrystal synthesis and infusion provides exquisite control of the nanocrystal size, morphology, and dispersibility within the pores. A dispersion of iridium nanocrystals was infused into mesoporous silica by expanding the solvent toluene with supercritical CO2. To achieve high nanocrystal loadings, up to 1.3 wt %, we tuned the solvent quality to strengthen the interactions of the nanocrystals with the pore walls, but without precipitating the nanocrystals in the bulk solvent. Z-contrast STEM indicates conclusively that the iridium nanocrystals were located within the pores and not on the external silica surface. High catalytic activity was observed for 1-decene hydrogenation, which is consistent with a high degree of dispersion of the 4.5 nm nanocrystals throughout the pores, as observed by TEM. A maximum turnover frequency (TOF) of 16 s(-1) was measured, which was higher than the initial TOF for homogeneous catalysis with the same nanocrystals in 1-decene. The iridium catalysts do not require pretreatment to remove the tetraoctylammonium bromide ligands to achieve activation, as the ligands bind weakly to the iridium surface. Consequently, the activity was not enhanced when calcined at 500 degrees C in nitrogen or when annealed in supercritical CO2 at 275 bar. The ability to predesign nanocrystal morphology and surface properties prior to infusion into the mesoporous silica support offers novel opportunities for enhanced catalyst activity, stability, and reaction selectivity.
The effects of solvent density on interparticle interactions between dispersed core-shell silica nanoparticles in liquid CO(2) were investigated in terms of diffusional second virial coefficients measured by dynamic light scattering. A porous cross-linked polymeric shell was formed about a hydrophilic silica core with a trifunctional silylating agent, (heptadecafluoro-1,1,2,2-tetrahydrodecyl) triethoxy silane. The addition of the porous polymeric shell weakened the Hamaker interactions between particles enabling dispersibility at low CO(2) pressures, even as low as the vapor pressure, with only a stir bar. With dynamic light scattering, the particle size and degree of aggregation were measured in ethanol, the solvent used for synthesis of the shells, and in CO(2) as a function of both the silane: silica (wt: wt) ratio and the silane addition rate. Particles in ethanol with thick shells could be dried and redispersed in CO(2), whereas particles with thin shells were dispersible in CO(2) only when added to CO(2) while still dispersed in ethanol. At the highest pressures, the diffusional second virial coefficients were only slightly negative in CO(2), indicating weak attractive interactions. The coefficients became more negative as the CO(2) density was lowered. Inorganic colloidal particles, with diameters on the order of a few hundred nanometers, may be dispersed in CO(2) at the vapor pressure for tens of minutes with porous crosslinked polymeric shells, whereas such stabilization has not been achievable with either non-cross-linked polymeric or low molecular weight stabilizers.
Aerosolized evaporative precipitation into aqueous solution and spray freezing into liquid nanostructured formulations of itraconazole as prophylaxis significantly improved survival relative to commercial itraconazole oral solution and the control in a murine model of invasive pulmonary aspergillosis. Aerosolized administration of nanostructured formulations also achieved high lung tissue concentrations while limiting systemic exposure.
Biopharmaceutics Classification System (BCS) Class II drugs are classified as having poor water solubility and good permeability across biological membranes. Improving the dissolution should, therefore, increase the bioavailability of this class of drugs. The Spray-Freezing into Liquid (SFL) process has been shown to increase dissolution rates of BCS class II drugs by providing amorphous particles with high surface area and small primary particle size, stabilized using hydrophilic polymers. Modifying the physicochemical properties of the particles is accomplished by selecting the optimum feed system composition. This article reviews the influence of this composition and active pharmaceutical ingredient (API) potency on surface area, surface morphology, crystallinity, and dissolution rates of model API.
The objective of this study was to investigate the influence of stabilizer type on the physicochemical properties, including dissolution, of ultra-high potency powders containing itraconazole (ITZ) formed by evaporative precipitation into aqueous Solution (EPAS). ITZ was dissolved in dichloromethane, which was then atomized through a heated coil at 80 degrees C into an aqueous solution over precise periods of time. Stabilizers were present in either the aqueous, organic or both phases. The dispersions were centrifuged and the supernatant was removed. Three hydrophilic stabilizers were investigated, including polysorbate 80, polyvinyl pyrrolidone and poloxamer 407. Rapid dissolving ultra-high potency of ITZ powders was successfully produced. Greater than 80% of ITZ was dissolved in 5 min compared to only 13% of ITZ bulk powders. The resulting stabilizer-coated drug particles had high drug-to-stabilizer ratios greater than 12, corresponding to potencies (wt drug/wt drug + wt surfactant) as high as 93%. An increase in dissolution rate was correlated with the amount of stabilizer adsorbed and the wettabilily. The combination of polysorbate 80 and poloxamer 407 present in the aqueous and organic phases, respectively, was superior in achieving high wetting and rapid dissolving ITZ powders. The ability to control the adsorption behavior of stabilizers by using synergistic combinations affords the opportunity to achieve high dissolution rates with higher potencies compared to previously reported values. (c) 2005 Elsevier B.V. All rights reserved.
We report electrostatic stabilization of micrometer-sized TiO2 particles at long range (several micrometers) in liquid and supercritical CO2 despite the ultralow dielectric constant, as low as 1.5. The counterions were solubilized in dry reverse micelles, formed with a low-molecular weight cationic perfluoropolyether trimethylammonium acetate surfactant, to prevent ion pairing with the particle surface. Dynamic light scattering and settling velocities indicate a particle diameter of 620-740 nm. The electrophoretic mobility of -2.3 x 10(-8) m(2)/V s indicated a particle charge on the order of -1.7 x 10(-17) C, or 105 elementary negative charges per particle. The balance of particle compression by an electric field versus electrostatic repulsion generated an amorphous arrangement of particles with 5-9 mu m spacing, indicating Debye lengths greater than 1 mu m. Scattering patterns also indicate that chains of particles may be achieved in CO2 by dielectrophoresis with alternating fields. The electrostatic stabilization has been achieved by solubilizing a small concentration of counterions in only a small fraction of the reverse micelles in the double layer. Whereas many low-molecular weight surfactants have been shown to form reverse micelles in CO2, very few polymers are able to stabilize micrometer-sized colloids sterically. Thus, electrostatic stabilization has the potential to expand markedly the domain of colloid science in apolar supercritical fluids.
A simple method to prepare surfactant-free and solvent-free semiconducting polymer particles by using an environmentally benign supercritical carbon dioxide (scCO(2)) process is reported. The process of the rapid expansion of supercritical solutions (RESS) is used to produce spherical particles of poly [2-(3-thienyl)acetyl 3,3,4,4,5,5,6,6,7,7,8, 8,8-tridecafluorooctanoate] (PSFTE), 50-500 nm in size, from 0.1-0.5 wt.-% PSFTE solutions in CO2 at pre-expansion temperatures of 40 degrees C and pre-expansion pressures of 276 bar.
The objective of this study was to compare the properties of particles formed by nucleation and polymer stabilization (e.g. evaporative precipitation into aqueous solution (EPAS)) versus rapid freezing (e.g. spray freezing into liquid (SFL)). Powders formed by EPAS and SFL, composed of danazol and PVP K-15 in a 1:1 ratio, were characterized using X-ray powder diffraction, modulated differential scanning calorimetry (MDSC), contact angle determination, dissolution, scanning electron microscopy (SEM), environmental scanning electron microscopy (ESEM), BET specific surface area, and Z-contrast scanning transmission electron microscopy (STEM). Large differences in particle morphologies and properties were observed and explained in terms of the particle formation mechanisms. Both techniques produced amorphous powders with high T-g and low contact angle values. However, STEM analysis showed highly porous bicontinuous nanostructured 30 nm particles connected by narrow bridges for SFL versus aggregated 500 nm primary particles for EPAS. The combination of STEM and other characterization techniques indicates solid solutions were formed for the SFL powders consistent with rapid freezing. In contrast, the EPAS particle cores are enriched in hydrophobic API and the outer surface is enriched in the hydrophilic polymer, with less miscibility than in the SFL powders. Consequently, dissolution rates are faster for the SFL particles, although both techniques enhanced dissolution rates of the API. (c) 2005 Elsevier B.V. All rights reserved.
high-pressure pendant-drop tensiometry, the interfacial tension (gamma) and surface excess (Gamma(infinity)) for a family of ionic surfactants with identical phosphate headgroups and varying fluorocarbon and hydrocarbon tail structures were examined at the water-CO2 interface. To compensate for the unusually weak CO2-surfactant tail interactions, we designed hydrocarbon tails with weak tail-tail interactions to achieve a more favorable hydrophilic-CO2-philic balance. Branching of hydrocarbon surfactant tails is shown to lead to more favorable adsorption at the interface, closer to that of fluorocarbon surfactants. gamma for a double-tail hydrocarbon phosphate surfactant with a relatively high degree of tail branching was lowered from the water-CO2 binary interface value of about 20 mN/m at 25 degreesC and 340 bar to 3.7 mN/m. This reduction in gamma is attributed to both a decrease in the free volume between tails at the interface and reduced tail-tail interactions. In addition to tail structure, the effects of surfactant counterion, salt concentration, temperature, and CO2 density on gamma and Gamma(infinity) were investigated. The hydrophilic-CO2-philic balances of these surfactants are mapped by investigating changes in interfacial tension with these formulation variables. Low-molecular-weight branched hydrocarbon ionic surfactants are shown to stabilize concentrated CO2-in-water emulsions for greater than 1 h.
Over the past decade, steric stabilization has been achieved for a variety of inorganic and organic colloids. in supercritical fluid carbon dioxide (SCCO2). Herein we demonstrate that colloids may also be stabilized in CO2 by electrostatic forces, despite the ultralow dielectric constant of 1.5. Zeta potentials of micrometersized water droplets, measured in a microelectrophoresis cell reached -70 mV corresponding to a few elementary charges per square micrometer of droplet surface. This degree of charge Was sufficient to stabilize water/CO2 emulsions for an hour, even with water volume fractions of 5%. Hydrogenions partition preferentially, relative to bicarbonate ions, from the emulsion droplet to,the cores-of surfactant micelles in the diffuse double layer surrounding the droplets. The micelles, formed with a low molecular weight branched hydrocarbon surfactant, prevent ion pairing of the hydrogen counterions to the negatively charged emulsion droplets. Dielectrophoresis of the water droplets at a frequency of 60 Hz leads to chains containing a dozen droplets with lengths of 50 mu m. The ability to form electrostatically stabilized colloids in carbon dioxide, is particularly useful in practical applications, because steric stabilization in CO2 is often limited by the poor solvation of the stabilizers.
Germanium nanocrystals were synthesized in supercritical (SC) CO2 by thermolysis of diphenylgermane (DPG) or tetraethylgermane (TEG) with octanol as a capping ligand at 500 degrees C and 27.6 MPa. The Ge nanocrystals were characterized with high resolution transmission electron microscopy (HRTEM), energy-dispersive x-ray spectroscopy (EDS), and x-ray diffraction (XRD). On the basis of TEM, the mean diameters of the nanocrystals made from DPG and TEG were 10.1 and 5.6 nm, respectively. The synthesis in sc-CO2 produced much less organic contamination compared with similar reactions in organic supercritical fluids. When the same reaction of DPG with octanol was performed in the gas phase without CO2 present, bulk Ge crystals were formed instead of nanocrystals. Thus, the solvation of the hydrocarbon ligands by CO2 was sufficient to provide steric stabilization. The presence of steric stabilization in CO2 at a reduced temperature of 2.5, with a reduced solvent density of only 0.4, may be attributed to a reduction in the differences between ligand-ligand interactions and ligand-CO2 interactions relative to thermal energy.
Stable protein nanostructured particles, produced by spray freezing into liquid (SFL) nitrogen, were encapsulated uniformly into microspheres to reduce the burst release over the first 24 h. The denaturation and aggregation of these bovine serum albumin (BSA) high-surface area particles were minimal due to ultra-rapid freezing and the absence of a liquid-air interface. Upon sonication, these friable highly porous, solid protein particle aggregates broke up into submicron particles. These particles were encapsulated into DL-lactide/glycolide copolymer (PLGA) and poly(lactic acid) (PLA) microspheres by anhydrous solid-in-oil-in-oil (s/o/o) techniques. For 5% loading of protein, the burst release after 24 h was only 2.5-4.1%, that is, values fivefold to tenfold lower than those observed for larger more conventional BSA particles. At a loading of 10%, the burst was only 6 and 13% for PLGA and PLA, respectively, and at 15% loading it was only 12% for PLGA. As shown with confocal and scanning electron microscopy (SEM), the low burst is consistent with a uniform distribution of protein nanoparticles, which were about 100 times smaller than the microspheres. Changes in aggregation and secondary structure, which were monitored by size exclusion chromatography and FTIR, respectively, indicated only slight monomer loss (3.9%) and high structural integrity (38% alpha-helix) in the encapsulated protein. (C) 2004 Wiley-Liss, Inc.
Steady-state fluorescence measurements have been used to measure the rate of transport of a fluorescent probe (pyrene) out of ca. 200-340 nm thick films of poly(methyl methaerylate) (PMMA) in contact with supercritical CO2 at pressures in the range 34-76 bar (estimated CO2 content in the film from 0.06 to 0.17 weight fraction) and several temperatures (35, 50, and 65 degreesC). At constant temperature, the estimated pyrene diffusion coefficient increases by approximately 4 orders of magnitude from the lowest to the highest CO2 content (e.g., from ca. 5 x 10(-15) cm(2)/S for ca. 0.08 CO2 weight fraction to ca. 10(-1)0 cm(2)/s for ca. 0.17 CO2 weight fraction at 35degreesC). We compare the present results to our earlier study of CO2-swollen polystyrene (PS) and find: (1) For similar pressures Of CO2 at the same temperature, the enhancement of the pyrene diffusion coefficient is larger in PMMA than in PS, presumably as a consequence of the higher solubility Of CO2 in PMMA; and (2) at similar CO2 contents, the pyrene diffusion coefficient is higher in PS than in PMMA by several orders of magnitude, which we attribute to the PS higher free volume for pyrene diffusion compared to PMXIA for CO2-swollen films.
Gold nanocrystal dispersions in toluene-CO2 Mixtures were infused into cylindrical pores in mesoporous silica to achieve high loadings over 2 wt % in 24 h. The nanocrystals were highly dispersed according to transmission electron microscopy, and the loadings approached equilibrium. In contrast, the loadings were small for infusion with pure toluene or toluene mixed with an antisolvent, methanol. The differences in loading were correlated with the long-ranged van der Waals forces between gold and silica through the intervening solvent. These van der Waals forces became stronger as CO2 was added to toluene, as a consequence of a reduction in the Hamaker constant of the mixed intervening solvent, resulting in stronger nanocrystal adsorption. The decoupling of the nanocrystal synthesis step and the infusion step leads to exquisite control of the nanocrystal size, morphology, and dispersibility within the pores. The simplicity of the method allows for the facile production of nanocrystal/silica composites for applications such as catalysis and optoelectronics.
High chemical yields, up to 73%, were achieved for germanium (Ge) nanocrystals synthesized in solution with germanium diiodide (GeI(1)2) and LiAlH4 as a reducing agent in tri-n-octylphosphine (TOP) or tri-n-butylphosphine (TBP). Ge nanocrystals were characterized by transmission electron microscopy (TEM), energy-dispersive X-ray spectroscopy (EDS), X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, and X-ray photoelectron spectroscopy (XPS). Reactions in TOP at 300 degrees C yielded Ge nanocrystals with moderate size polydispersity and good crystallinity with average diameters that could be manipulated by varying the precursor concentration from similar to 3 to similar to 11 nm. High chemical yields are enabled by the high reactivity of GeI2, high GeI2 solubility in alkyl phosphines, and relatively mild reaction temperature, which minimizes byproduct formation and solvent degradation. Compared to reactions carried out in TOP with the same concentration of GeI2, nanocrystals synthesized in TBP at 240 degrees C exhibit larger size and broader size distribution. The presence of alkyl groups in the FTIR spectra, the small and controllable particle diameters, and a lack of significant Ge oxidation revealed by XPS indicate that the nanocrystals were chemically passivated with an organic layer.
The aim of research was to design a small, restraint free, low stress animal dosing chamber for inhalation studies, and to investigate distribution of a model drug within the chamber. A small animal dosing chamber was designed that consisted of a polymethylmethacrylate (MMA) airtight box (40.6 x 11.4 x 21.6 cm) with a hinged top, having a nominal wall thickness of 1.25 cm. The chamber was designed to hold up to 14 mice. each having a floor area of approximately 63 cm(2), in accordance with Institutional Animal Care and Use Committee (IACUC) guidelines. A "rodent proof" distribution fan was attached to the center of the hinged closure lid. The chamber was divided into 1 inch zones (120 in total) to enable a profile of drug distribution within the chamber to be obtained. Small holes were drilled into the side of the chamber and sealed using Parafilm((R)) to allow access to the sampling zones. Syringes (5 mL) with appropriate length poly-tetrafluoroethylene (PTFE) tubing were inserted into the holes to reach the sampling zones (eight on either side of the chamber giving a total of 16 zones). An aqueous caffeine solution (2% w/v) in glycerol (25% w/v) was prepared and nebulized into the chamber using an Aeroneb Pro((R)) nebulizer. Caffeine containing droplets were circulated into the chamber at a flow rate of 1.5 L/min(-1), and the air was recirculated in a closed system for a total of 20 minutes to ensure a high concentration of caffeine droplets throughout. Following nebulization, air samples (5 mL) were withdrawn from the 16 sampling zones of the sealed chamber. The process was repeated in quadruplet until a total of 64 sampling zones had been sampled. The entire experiment was also repeated with the absence of the "rodent-proof" distribution fan. Drug concentrations were calculated from a calibration curve of caffeine using UV absorbance at 272 nm. An average mass of caffeine (Standard Deviation; S.D.) of 5.0 (4.2) mg was detected throughout the chamber when the distribution fan was fitted, and caffeine 12.6 (9.7) mg was detected without the fan. This indicated that presence of the fan caused impingement of the drug on both the chamber walls and fan components; effectively removing nebulized drug from circulation within the chamber. The distribution of drug was plotted using a 3D graph: this revealed a lower concentration at the periphery and a higher concentration in the center of the chamber both with and without the distribution fan in place. In conclusion, a humane, nonrestraint rodent closing chamber was designed for the efficient delivery of nebulized drugs for up to 14 mice simultaneously. The highest levels of the model drug caffeine were detectable throughout the small animal dosing chamber without the distribution fan. A circulation flow rate of 1.5 L/min(-1) was found to be adequate to distribute drug in the chamber. Surprisingly, the results demonstrate that avoiding the use of a distribution fan altogether maximizes the drug concentration within the chamber by reducing impingement of the nebulized drug. The small animal, restraint-free dosing chamber represents an advancement in reproducible dosing via the pulmonary route in the small animal model. The dosing chamber may be adapted to present the lung with an almost unlimited array of compounds, encompassing drugs, toxic compounds, and even pathogens, while still maintaining a relatively stress-free microenvironment for the test subject and furthermore, total safety for the operator.