Precipitation With A Compressed Antisolvent Research Articles

High Loading of Gentamicin in Bioadhesive PVM/MA Nanostructured Microparticles Using Compressed Carbon-Dioxide

To investigate, for the first time, the viability of compressed antisolvent methodologies for the preparation of drug-loaded particles of the biodegradable and bioadhesive polymer poly (methyl vinyl ether-co-maleic anhydride) (PVM/MA), utilizing gentamicin (Gm) as a model drug. Precipitation with a Compressed Antisolvent (PCA) method was used for the preparation of PVM/MA particles loaded with gentamicin. Before encapsulation, gentamicin was modified into a hydrophobic complex, GmAOT, by exchanging its sulphate ions with an anionic surfactant. GmAOT:PVM/MA composites were fully characterized in terms of size, morphology, composition, drug distribution, phase composition, in vitro activity and drug release. Homogeneous nanostructured microparticles of PVM/MA loaded with high and uniformly distributed quantities of GmAOT were obtained by PCA. The drug loading factors could be tuned at will, improving up to ten times the loadings obtained by other precipitation techniques. Gentamicin retained its bioactivity after being processed, and, according to its release profiles, after an initial burst it experienced a sustained release over 30 days. Compressed antisolvent methods are suitable technologies for the one-step preparation of highly loaded nanostructured PVM/MA matrices with promising application in the delivery of low bioavailable drugs.

Novel bioactive hydrophobic gentamicin carriers for the treatment of intracellular bacterial infections

Gentamicin (GEN) is an aminoglycoside antibiotic with a potent antibacterial activity against a wide variety of bacteria. However, its poor cellular penetration limits its use in the treatment of infections caused by intracellular pathogens. One potential strategy to overcome this problem is the use of particulate carriers that can target the intracellular sites of infection. In this study GEN was ion-paired with the anionic AOT surfactant to obtain a hydrophobic complex (GEN–AOT) that was formulated as a particulated material either by the precipitation with a compressed antisolvent (PCA) method or by encapsulation into poly(d,l-lactide-co-glycolide) (PLGA) nanoparticles (NPs). The micronization of GEN–AOT by PCA yielded a particulated material with a higher surface area than the non-precipitated complex, while PLGA NPs within a size range of 250–330nm and a sustained release of the drug over 70days were obtained by preparing the NPs using the emulsion solvent evaporation method. For the first time, GEN encapsulation efficiency values of ∼100% were achieved for the different NP formulations with no signs of interaction between the drug and the polymer. Finally, in vitro studies against the intracellular bacteria Brucella melitensis, used as a model of intracellular pathogen, demonstrated that the bactericidal activity of GEN was unmodified after ion-pairing, precipitation or encapsulation into NPs. These results encourage their use for treatment for infections caused by GEN-sensitive intracellular bacteria.

Preparation of polystyrene/poly(methyl methacrylate) blends by compressed fluid antisolvent technique

Submicron polystyrene (PS)/poly(methyl methacrylate) (PMMA) blends were generated by the precipitation with a compressed antisolvent (PCA) technique. The generation of PS/PMMA blends was carried out by spraying a solution containing PS and PMMA into a precipitator. The blends without coalescence were observed to only be generated when both vapor and liquid CO 2 existed in the precipitator combined with appropriate total polymer concentration in solution, molecular weights (Mws) of PS and PMMA, mass ratio of PS to PMMA, flow rates of CO 2 and polymer solution, and liquid CO 2 level in the precipitator. Two Mws of PS, 144,000 and 44,000, and two Mws of PMMA, 85,000 and 36,000, were used in this study. It was found that the blends could be easier to generate using a higher PS Mw, a lower PMMA Mw, and a higher mass ratio of PS to PMMA. Toluene with a solubility parameter smaller than that of tetrahydrofuran (THF) was found to be the more appropriate solvent for generating spherical PS/PMMA submicron blends. The SEM and TEM images show that the spherical PS/PMMA core/shell blends could be generated at a temperature of 298 K, a pressure of 6.41 MPa, a liquid CO 2 level of 1/2 of the precipitator, a CO 2 flow rate of 2000 mL/min, a solution flow rate of 5 or 10 mL/min, and a total polymer concentration of 0.72 wt% for a PS Mw of 144,000, a PMMA Mw of 36,000, and a PS/PMMA mass ratio of 9/1. Individual and spherical PS and PMMA particles or spherical PS particles partially covered by a PMMA films, however, were generated when the liquid CO 2 level was of 1/8 or lower in the precipitator. A possible mechanism for the formation of core–shell blend was proposed.

Compressed CO 2 antisolvent precipitation of lysozyme

The precipitation of proteins using compressed CO 2 as antisolvent in the precipitation with a compressed antisolvent (PCA) process has been investigated for lysozyme in its DMSO solution. In the vapor liquid coexistence region of the solvent–antisolvent mixture, particles between 100 and 200 nm were obtained, whereas at supercritical and liquid conditions studied in the 25–40 ° C and 100–150 bar ranges, particle sizes were reproducibly below 100 nm. No effect on particle size was observed for variations of the CO 2 and solutions flow rates between 40 and 120 g/min, and between 0.4 and 2 ml/min, respectively. Phase behavior experiments indicate that protein precipitation results from a metastable liquid–liquid phase separation, with the protein rich phase remaining trapped in a gel-like state. Moreover, we suggest that due to the high CO 2 concentrations involved in PCA, phase separation takes place by spinodal decomposition. The proposed precipitation mechanism is not limited to the PCA process, but can be extended also to results obtained for the precipitation of lysozyme using the gas antisolvent (GAS) process.

Modelling mass transfer in the PCA process using the Maxwell–Stefan approach

The two-way mass transfer between a dichloromethane droplet and carbon dioxide at the typical conditions of the Precipitation with a Compressed Antisolvent (PCA) process has been modelled using a finite-difference approximation of the Maxwell–Stefan equations. The developed model analyses the variation of concentration, fluxes at the droplet interface and droplet diameter with time, and predicts how different operating conditions will affect droplet size and droplet evaporation time. Simulations show that in the studied pressure and temperature range (4–7.5 MPa and 308–328 K) absorption of CO 2 into the liquid phase is always faster than solvent evaporation. Droplets produced as result of atomisation in the nozzle will swell as soon as they get into contact with CO 2. Droplet lifetime can be reduced by an increase of the CO 2 to solvent molar ratio and by a decrease of the initial droplet diameter. The different factors enhancing/slowing down mass transfer have a complex dependence on pressure and temperature. At high pressures and low temperatures droplet lifetime is long due to the larger diameter attained by the droplets as result of the high solubility of carbon dioxide in the liquid phase; at very low pressures and high temperatures droplet lifetime increases due to the lower density of the vapour phase.

Microparticle-based lung delivery of INH decreases INH metabolism and targets alveolar macrophages

Microparticles prepared by the precipitation with a compressed antisolvent (PCA) process were evaluated for their potential in targeting an ionizable prodrug of isoniazid (INH), isoniazid methanesulfonate (INHMS), for sustained delivery of INH to alveolar macrophages (AMs). The charged prodrug was ion-paired with two different hydrophobic cations (tetrapentylammonium (TPA)— and tetraheptylammonium (THA)—bromide), and loaded separately into the poly( l-lactide) (PLA) microparticles. The drug/polymer particles were spherical in shape and between 1 and 3 μm in diameter. The choice of hydrophobic cations did not affect drug incorporation efficiencies or the release kinetics of INH from the microparticles. Using a sensitive liquid chromatographic tandem mass spectrometric (LC-MS/MS) assay developed for INH, high level of INH was detected in NR8383, a rat AM cell line, following exposure of these cells to drug-loaded microparticles. To confirm the microparticles can target AMs in vivo, we compared the INH levels in lavaged bronchoalveolar macrophages by LC-MS/MS after the Sprague–Dawley rats were administered either INHMS in PLA microparticles by intra-tracheal instillation or INH solution by gavage or intra-tracheal instillation. As expected, only microparticles provided sustained and targeted delivery of INH to AMs. Most importantly, this method of delivery led to substantial reduction in the blood levels of acetylisoniazid (AcINH), a major and potential toxic metabolite of INH.

Operating regimes and mechanism of particle formation during the precipitation of polymers using the PCA process

The vapour–liquid equilibrium phase behavior of a solvent and carbon dioxide provides two different regions of operation for the precipitation with a compressed antisolvent (PCA) process. Below the critical pressure of the mixture there is an interface between the liquid- and vapour-phase. Solution droplets are formed by atomisation in the nozzle. Above the critical- (or saturation-) pressure there is no phase boundary and contact between CO 2 and solution takes place by mixing. Additionally, in both operating regions, droplets of a polymer rich-phase are formed as result of a liquid–liquid phase split induced in the polymer solution when it gets in contact with the CO 2. This article provides experimental evidence for the hypothesis that when processing polymer solutions there are two different mechanisms of droplet formation governing the final size of the precipitated polymer particles: hydraulic atomisation and liquid–liquid phase split. The system l-polylactic acid ( l-PLA)–dichloromethane (DCM)–CO 2 was used to demonstrate that particle size can be manipulated by modifying the operating conditions. Working at conditions below the mixture critical pressure of the solvent–CO 2 mixture it was possible to produce polymer particles in the range of 5–50 μm. At conditions where the two fluids are completely miscible, l-PLA particles ranging from 0.1 to 2 μm and microfibers were obtained. The possibility of co-precipitation of cholesterol and l-PLA was also addressed.

Degradation kinetics of high molecular weight poly(L-lactide) microspheres and release mechanism of lipid:DNA complexes

Plasmid DNA encoding the green lantern protein was ion-paired with 1,2-dioleoyl, 3-trimethylammonium propane (DOTAP) at a (+/−) charge ratio of (1:1) to form a hydrophobic ion-pair (HIP) complex using the Bligh and Dyer method, and transferred into methylene chloride. Precipitation with a compressed antisolvent (PCA) was then employed to encapsulate plasmid DNA into poly(L-lactide) (PLLA) microspheres. The hydrophobicity of DOTAP:DNA complexes allowed consistently high encapsulation efficiencies (>70%) to be achieved. Release of the DOTAP:DNA complex from PLLA microspheres exhibitedminimal burst and a short (ca. 1 week) lag phase, followed by sustained release over a 20 week period. Release kinetics were consistent with a simple Fickian diffusion model. No correlation was identified between release rate of soluble poly(L-lactide) species (≤10 lactate units) from PLLA and the DNA release kinetics. Only ~12% of the polymer was degraded into soluble poly(L-lactide) over the time frame where ~90% of the plasmid load had been released.

Improved PCA process for the production of nano‐ and microparticles of polymers

AbstractThe system dextran‐DMSO‐CO2 has been chosen as a model system to study the fundamentals of the precipitation of biodegradable polymers, using the precipitation with a compressed antisolvent (PCA) process. At conditions of completely miscibility between the organic solvent (DMSO) and CO2, it is proposed that the formation of droplets in the system is not the consequence of atomization of the DMSO solution, but rather the result of the occurrence of a liquid‐liquid phase split when the DMSO‐solution and CO2 are intimately mixed. Penetration of CO2 into the droplets and stripping of DMSO induces the solidification of these droplets. A new device was designed to separate the three steps involved in the process: mixing of CO2 and solution, liquid‐ liquid phase split, and stripping of the solvent. This new device eliminates agglomeration of particles, and yields reproducible results. The particle size can be easily manipulated over a size range from several nanometers to tenths of microns by changes in the operating conditions. The influence of these operating conditions (pressure, temperature, polymer concentration, and solution/CO2 composition ratio) on particle morphology has been studied. © 2004 American Institute of Chemical Engineers AIChE J, 50: 2408–2417, 2004

Preparation of budesonide and budesonide-PLA microparticles using supercritical fluid precipitation technology

The objective of this study was to prepare and characterize microparticles of budesonide alone and budesonide and polylactic acid (PLA) using supercritical fluid (SCF) technology. A precipitation with a compressed antisolvent (PCA) technique employing supercritical CO2 and a nozzle with 100-μm internal diameter was used to prepare microparticles of budesonide and budesonide-PLA. The effect of various operating variables (temperature and pressure of CO2 and flow rates of drug-polymer solution and/or CO2) and formulation variables (0.25%, 0.5%, and 1% budesonide in methylene chloride) on the morphology and size distribution of the microparticles was determined using scanning electron microscopy. In addition, budesonide-PLA particles were characterized for their surface charge and drug-polymer interactions using a zeta meter and differential scanning calorimetry (DSC), respectively. Furthermore, in vitro budesonide release from budesonide-PLA microparticles was determined at 37°C. Using the PCA process, budesonide and budesonide-PLA microparticles with mean diameters of 1 to 2 μm were prepared. An increase in budesonide concentration (0.25%–1% wt/vol) resulted in budesonide microparticles that were fairly spherical and less aggiomerated. In addition, the size of the microparticles increased with an increase in the drug-polymer solution flow rate (1.4–4.7 mL/min) or with a decrease in the CO2 flow rate (50–10 mL/min). Budesonide-PLA microparticles had a drug loading of 7.94%, equivalent to ∼80% encapsulation efficiency. Budesonide-PLA microparticles had a zeta potential of— 37±4 mV, and DSC studies indicated that SCF processing of budesonide-PLA microparticles resulted in the loss of budesonide crystallinity. Finally, in vitro drug release studies at 37°C indicated 50% budesonide release from the budesonide-PLA microparticles at the end of 28 days. Thus, the PCA process was successful in producing budesonide and budesonide-PLA microparticles. In addition, budesonide-PLA microparticles sustained budesonide release for 4 weeks.

Preparation of budesonide and budesonide-PLA microparticles using supercritical fluid precipitation technology.

The objective of this study was to prepare and characterize microparticles of budesonide alone and budesonide and polylactic acid (PLA) using supercritical fluid (SCF) technology. A precipitation with a compressed antisolvent (PCA) technique employing supercritical CO2 and a nozzle with 100- microm internal diameter was used to prepare microparticles of budesonide and budesonide-PLA. The effect of various operating variables (temperature and pressure of CO2 and flow rates of drug-polymer solution and/or CO2) and formulation variables (0.25%, 0.5%, and 1% budesonide in methylene chloride) on the morphology and size distribution of the microparticles was determined using scanning electron microscopy. In addition, budesonide-PLA particles were characterized for their surface charge and drug-polymer interactions using a zeta meter and differential scanning calorimetry (DSC), respectively. Furthermore, in vitro budesonide release from budesonide-PLA microparticles was determined at 37 degrees C. Using the PCA process, budesonide and budesonide-PLA microparticles with mean diameters of 1 to 2 microm were prepared. An increase in budesonide concentration (0.25%-1% wt/vol) resulted in budesonide microparticles that were fairly spherical and less agglomerated. In addition, the size of the microparticles increased with an increase in the drug-polymer solution flow rate (1.4-4.7 mL/min) or with a decrease in the CO2 flow rate (50-10 mL/min). Budesonide-PLA microparticles had a drug loading of 7.94%, equivalent to approximately 80% encapsulation efficiency. Budesonide-PLA microparticles had a zeta potential of -37 +/- 4 mV, and DSC studies indicated that SCF processing of budesonide-PLA microparticles resulted in the loss of budesonide crystallinity. Finally, in vitro drug release studies at 37 degrees C indicated 50% budesonide release from the budesonide-PLA microparticles at the end of 28 days. Thus, the PCA process was successful in producing budesonide and budesonide-PLA microparticles. In addition, budesonide-PLA microparticles sustained budesonide release for 4 weeks.

Formation of Nylon Particles and Fibers Using Precipitation with a Compressed Antisolvent

Micrometer-sized particles and fibers of nylon 6/6 were produced by the means of precipitation with a compressed antisolvent (PCA). Specifically, nylon 6/6 micron-sized particles were produced by expanding a nylon/formic acid solution through various capillary nozzles into compressed carbon dioxide antisolvent. The effect of various operating parameters, such as the nylon/formic acid solution concentration, nozzle length and diameter, and nylon solution injection velocity, were examined, and no significant effect of varying operating conditions in the mean particle size, and particle size distribution was observed. In almost all of the cases, spherical particles were obtained. However, under certain circumstances, oblong particles (short fibers), fibers, and thin film morphologies were observed by manipulating various process parameters, including nylon solution concentration and the manner of spraying the solvent/nylon solution into the carbon dioxide antisolvent. The lack of significant influence of the varying operating conditions on the mean size may be explained in that the surface tensions of the sprayed droplets diminish at very short distances from the nozzle outlet as compared to the jet breakup length. In other words, the disappearance of surface tension stimulates gaslike mixing, without the formations of discrete droplets.

Alveolar macrophage cell line is not activated by exposure to polymeric microspheres

An in vitro cell culture model based on a rat alveolar macrophage (AM) cell line, NR8383, was used to determine if poly( l-lactide) (PLA) microspheres prepared by the precipitation with a compressed antisolvent (PCA) method can be taken up by AMs and activate AMs. To examine cellular uptake of microspheres, microspheres were labeled with rhodamine 6G. Using fluorescence microscopy, the uptake of microspheres by NR8383 cells was followed as a function of time, microsphere concentration, and susceptibility to lysosomotropic agents. To determine if microspheres can activate NR8383 cells, the oxidative burst and production of TNF- α by NR8383 cells following microsphere treatment was measured. Uptake of microspheres by NR8383 cells was dependent on microsphere concentration and appeared to occur via endocytosis, as uptake was significantly inhibited by the putative lysosomotropic agents, ammonium chloride and chloroquine. Furthermore, the microspheres do not appear to activate NR8383 cells, since microsphere exposure results in negligible oxidative burst and TNF- α production in NR8383. Microspheres prepared by the PCA method hold great potential in targeting drugs to AMs and, therefore, may be of utility for the treatment of diseases in which AMs play an important role, such as tuberculosis (TB).

Process variable implications for residual solvent removal and polymer morphology in the formation of gentamycin-loaded poly (L-lactide) microparticles.

The purpose was to determine the influence of process parameters in the precipitation with a compressed antisolvent (PCA) process on the morphology and residual dichloromethane (DCM) levels in gentamycin-loaded PLA microparticles. The three variables studied were the rate of CO2 co-flowed during the polymer and drug post-precipitation, the post-precipitation pure CO2 flush rate, and the post-precipitation CO2 flush volume. Residual DCM levels were determined from headspace gas chromatography-mass spectroscopy (GC-MS) with single ion monitoring. X-ray diffraction (XRD) and differential scanning calorimetry (DSC) were used to estimate the crystallinity within microparticles. DCM was extracted from drug-loaded microparticles by both supercritical CO2 extraction and vacuum drying for up to two days to determine a lower limit for solvent removal. Increasing either the post-precipitations CO2 flow rate or flush volume resulted in lower residual DCM levels in the microparticle. The CO2 co-flow rate showed an opposite trend. Increasing in value resulted in a higher DCM value after precipitation. XRD and DSC analysis on these samples suggest that those produced at lower CO2 co-flow rates have a higher degree of crystallinity, which increases the diffusivity of DCM through the polymer matrix. Finally, samples subjected to extended (48 hr) CO2 extraction resulted in DCM levels on the order of one to three ppm. Specific PCA process conditions during microparticle formation have a strong influence on the residual solvent levels within the microparticles. Polymer morphology affects the diffusivity of solvent through the polymer matrix, which in turn determines the solvent removal rates.

Controlled release of ionic compounds from poly ( l-lactide) microspheres produced by precipitation with a compressed antisolvent

Poly ( l-lactide) microspheres containing low molecular weight pharmaceutical agents were prepared using the precipitation with a compressed antisolvent (PCA) process with supercritical carbon dioxide as the antisolvent. Gentamycin, naloxone, and naltrexone were solubilized in methylene chloride using hydrophobic ion pairing (HIP) to stoichiometrically replace polar counter ions with an anionic detergent, aerosol OT (AOT, sodium bis-2-ethylhexyl sulfosuccinate). Through HIP complexation, solubilities in excess of 1 mg/ml were attainable in methylene chloride, allowing levels of direct incorporation that are not possible with other PCA approaches. The drug/polymer particles were spherical in shape and between 0.2 and 1.0 μm in diameter, as determined by scanning electron microscopy. Drug incorporation efficiencies were determined and in vitro release profiles measured. At 37°C, the release of the ion-paired drugs into phosphate-buffered saline displays minimal burst effects and exhibits release kinetics that are approximately linear with the square root of time, indicating matrix diffusion control of drug release. For gentamycin, linear release from the poly ( l-lactide) microspheres was observed for more than 7 weeks, even at a drug loading of near 25% (w/w). Naltrexone exhibits similar release characteristics, although more drug was found on the surface of the microspheres. Conversely, rifampin, which was not ion-paired, was poorly encapsulated.