Supercritical assisted atomization: A novel technology for microparticles preparation of an asthma-controlling drug

The objective of this study was to produce microparticles of a new asthma-controlling drug by supercritical assisted atomization (SAA), proposed as an alternative to conventional jet-milling process. SAA is based on the solubilization of supercritical carbon dioxide in a liquid solution containing the drug; the ternary mixture is then sprayed through a nozzle, and microparticles are formed as a consequence of the enhanced atomization. SAA process parameters studied were precipitator temperature, nozzle diameter, and drug concentration in the liquid solution. Their influence was evaluated on morphology and size of precipitated particles. Spherical particles with mean particle size ranging from 1 to 3 μm of the new anti-asthma drug were produced by SAA. The mass median aerodynamic diameter (MMAD) of the SAA micronized particles and of the conventional jet-milled drug was used to compare, the results obtainable using the 2 techniques. Particularly, MMADs from 1.6 to 4.0 μm were obtained by SAA at the optimum operating conditions and by varying the concentration of the solution injected. MMAD of 6.0 μm was calculated for the jet-milled drug. SAA samples also exhibited narrower particle size distribution (PSD). A good control of particle size and distribution together with no drug degradation was obtained by SAA process. Published: October 22, 2005     

Cyclodextrins micrometric powders obtained by supercritical fluid processing

Supercritical fluid technology offers the possibility to produce dry powder formulations of biocompatible materials, overcoming the drawbacks of classical micronization processes. In this work, Supercritical Assisted Atomization (SAA) has been used to micronize alpha-cyclodextrin (alpha-CD) and hydroxypropyl-beta-cyclodextrin (HP-beta-CD). Some process parameters, such as precipitation temperature and solute concentration in the liquid solution, have been studied to evaluate their influence on morphology and size of precipitated particles. Cyclodextrins (CDs) micronization has been successful: well-defined spherical microparticles of alpha-CD and HP-beta-CD have been produced. Particle size analysis revealed that sharp distributions have been obtained: 95% of particles have diameters ranging between 0.1 and 5 microm for both CDs. X-ray and DSC analyses have been also performed to investigate CDs modifications induced by SAA processing: amorphous particles have been obtained in both cases, whereas raw alpha-CD was crystalline and raw HP-beta-CD was amorphous.     

Micronization of lysozyme by supercritical assisted atomization

Supercritical Assisted Atomization (SAA) has been used to produce lysozyme microparticles. Lysozyme has been micronized using water, buffered water at pH 6.2 and water–ethanol mixtures at different volume percentages. Precipitated lysozyme particles were spherical, with a narrow particle size distribution (PSD) ranging from 0.1 to 4 µm. The concentration of lysozyme in the liquid solvent mixture had a nonlinear effect on the particle distribution, with an increase of the X_0.9 from about 1 to 3 µm varying the enzyme concentration from 5 to 20 mg/mL. Precipitation temperature was set as low as possible to avoid enzyme degradation. High‐performance liquid chromatography analysis showed no degradation of lysozyme and the enzyme activity, measured by turbidimetric enzymatic assay, only slightly decreased after SAA processing. Depending on the process conditions lysozyme retained from 95% to 100% of the biological activity compared to the untreated enzyme. Biotechnol. Bioeng. 2009; 104: 1162–1170. © 2009 Wiley Periodicals, Inc.     

Micronization of Organic Materials by Crystallization with Compressed Gases

A pilot plant is presented, which has been built to prepare fine particles by the P recipitation with a C ompressed Fluid A ntisolvent (PCA process). This technique offers interesting applications for products, which are produced in small amounts, as certain pharmaceuticals or energetic materials. In this contribution the micronization of paracetamol and tartaric acid is presented. Liquid solutions of tartaric acid in acetone, ethanol and methanol/ethanol mixtures have been sprayed into supercritical carbon dioxide used as antisolvent. The mean particle size of the precipitated powder can be manipulated by changing the precipitation pressure and solvent type however the precipitation temperature has no significant influence on the particle size. Paracetamol is micronized from acetone, methanol and DMF and morphologies from needles to spheres were found depending on the solvent. The particle size was in the submicron range.     

Production of micronic particles of biocompatible polymer using supercritical carbon dioxide

Three micronization techniques, based on the use of supercritical carbon dioxide, were investigated to produce microspheres of a natural biocompatible polysaccharide. Particles smaller than 20 mum were obtained by means of the rapid expansion of a supercritical solution method (RESS), both with and without cosolvents. The mean diameter of the particles was reduced to about 0.5 mum when a solution of the polymer in an organic solvent was expanded by using carbon dioxide as a supercritical antisolvent (SAS). The SAS process was operated both in a continuous and in a batch mode. The former leads to aggregated structures and fibers, and the latter to the formation of micronic spherical particles. It was found that the experimental temperature did not substantially affect the shape and dimension of the particles. A stronger dependence is shown with respect to the solute concentration in the starting solution. The proposed method is attractive as the basis of a new process for the preparation of drug delivery systems. (c) 1997 John Wiley & Sons, Inc.     

Microparticles of soy lecithin formed by supercritical processes

Finely divided particles of phospholipids are used to form controlled drug delivery systems called liposomes. Conventional physicochemical methods for preparing these microparticles are hampered by a major drawback-the use of organic solvents that remain at few but inhibitory concentration in the final product. This study aimed to propose an alternative method for preparing microparticles of phospholipids starting from soy lecithin-the process had to be free of solvent or at least, the solvent had to be nontoxic. Two micronization techniques based on the use of supercritical carbon dioxide were investigated: the RESS and the SAS processes. The RESS process failed to separate the particles formed from the cosolvent. Performing the SAS process with ethanol as auxiliary solvent, enabled fine particles to form with size ranging from 1 to 40 microm. Particles were spherical and partly agglomerated and seemed to be free of solvent as shown by preliminary infrared analysis.     

Drug-polymer microparticles produced by supercritical assisted atomization

The supercritical assisted atomization (SAA) was proposed as a new technique to produce composite microparticles for drug controlled release. Ampicillin trihydrate and chitosan were selected as model drug and carrier, respectively, and 1% v/v acetic acid aqueous solution was used as solvent. The effect of the polymer/drug ratio on particle morphology and drug release rate was evaluated. SEM analysis indicated that non-coalescing spherical microparticles formed by chitosan/ampicillin were produced by SAA. All coprecipitates produced have a sharp particle distribution, with diameters ranging between about 0.1 and 6 microm. SAA composite microparticles were characterized by X-ray, DSC, EDX and UV-vis analysis. A solid solution of the chitosan and ampicillin was produced and a stabilizing effect of the polymer on the drug has resulted that protects ampicillin from thermal degradation. A prolonged release from SAA coprecipitates with respect to raw drug and physical mixtures of chitosan and ampicillin was obtained; moreover, the polymer/drug ratio has revealed to be a controlling parameter for drug release. Drug release mechanisms characteristic of swelling-controlled systems were observed, with ampicillin release depending on both relaxation and diffusive mechanisms. An empirical binomial equation was used to describe experimental data, showing a fair good agreement with ampicillin release data if both the relaxational and the diffusional parameters are function of the polymer/drug ratio.     

反溶剂重结晶法制备牡荆苷微粉工艺及其表征

为了提高牡荆苷溶出度,本实验采用反溶剂重结晶法（以N-甲基吡咯烷酮为溶剂,水为反溶剂）对牡荆苷原粉进行超细化研究。考察了药物浓度、溶剂与反溶剂体积比、搅拌转速及表面活性剂(PVP、Tween80、SDS)对牡荆苷微粉粒径的影响,确定牡荆苷微粉的最佳制备条件为：药液浓度为30 mg·mL^-1,反溶剂与溶剂体积比为15∶1,搅拌转速为1 500 r·min^-1,反溶剂中表面活性剂PVP浓度为8 mg·mL^-1,上述条件下制备的牡荆苷微粉平均粒径为291.1 nm;采用扫描电镜(SEM)、X射线衍射(XRD)、差示扫描(DSC)、红外光谱(FTIR)对牡荆苷原粉与微粉进行了表征,与原粉相比牡荆苷微粉粒径变小,结晶度降低,其化学性质未发生改变,体外溶出度显著提高。     

Vesicles mimicking normal and cancer cell membranes exhibit differential responses to the cell-penetrating peptide Pep-1

The cell-penetrating peptide (CPP) Pep-1 presents a great potential in drug delivery due to its intrinsic property to cross plasma membrane. However, its mechanism of entry into the cell remains unresolved. In this study, we compare the selectivity of Pep-1 towards vesicles mimicking normal and cancer cell membranes. The interaction was performed in a wide range of peptide-to-lipid molar ratios using infrared (IR), fluorescence, scanning electron microscopy (SEM), thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) techniques. At low peptide concentration, fluorescence experiments show that lipid-phosphatidylserine (PS) seems to enable Pep-1 translocation into cancer cell membrane as evidenced by the blue shift of its maximal emission wavelength. DSC data show that Pep-1 induces segregation of lipids. At high peptide concentration, IR data indicate that the interaction of Pep-1 is relatively stronger with normal cell membrane than with cancer cell membrane through the phosphate groups, while the interaction is weaker with normal cell membrane than with cancer cell membrane through the carbonyl groups. TGA and DSC data reveal that vesicles of normal cell membrane are thermally more stable than vesicles of cancer cell membrane. This suggests that the additional lipid PS included in cancer cell membrane has a destabilizing effect on the membrane structure. SEM images reveal that Pep-1 form superstructures including spherical particles and fibrils in the presence of both model membranes. PS seems to enhance peptide transport across cellular membranes. The biophysical techniques in this study provide valuable insights into the properties of CPPs in drug delivery systems.     

Formation of bioerodible polymeric microspheres and microparticles by rapid expansion of supercritical solutions.

Polyhydroxy acids [poly(L-lactic acid) (L-PLA), poly(D,L-lactic acid) (DL-PLA), and poly-(glycolic acid) (PGA)], biocompatible and bioerodible polymers that are being investigated for controlled delivery of pharmaceuticals and are approved by the Food and Drug Administration for in vivo sutures and bone repair implants, have been dissolved in supercritical CO2 and precipitated by rapid expansion of the resulting supercritical solutions (RESS). The formation of these microparticles and microspheres is a first step toward the goal of producing, in a single processing step, drug-loaded polymeric microspheres for use in controlled release applications. Nucleation of poly(L-lactic acid) from CO2 and CO2-acetone mixtures produced microparticles and microspheres ranging from 4 to 25 microns. Microspheres (2-20 microns) were also obtained with chlorotrifluoromethane as solvent. Commercial L-PLA precipitated after extraction of low molecular weight oligomers showed degradation kinetics similar to that of the starting material. The precipitation of DL-PLA from CO2 produced irregular-sized particles (10-20 microns). PGA, a polymer insoluble in most organic solvents, was found to be soluble in supercritical CO2. Nucleation of PGA from CO2 produced both regular-sized particles and needles of 10-40-microns length. The total solubility of commercial L-PLA in supercritical CO2 at 250 bar and 55 degrees C decreased from 0.14 wt % to less than 0.05 wt % and then leveled off as the cumulative flow of CO2 per unit mass of L-PLA loaded in the extractor increased beyond 20 standard L of CO2/g of L-PLA. Use of acetone (1 wt %) as a cosolvent increased L-PLA solubility by approximately 500%.     

Self-Associated Submicron IgG1 Particles for Pulmonary Delivery: Effects of Non-ionic Surfactants on Size, Shape, Stability, and Aerosol Performance

The ability to produce submicron particles of monoclonal antibodies of different sizes and shapes would enhance their application to pulmonary delivery. Although non-ionic surfactants are widely used as stabilizers in protein formulations, we hypothesized that non-ionic surfactants will affect the shape and size of submicron IgG particles manufactured through precipitation. Submicron particles of IgG1 were produced by a precipitation process which explores the fact that proteins have minimum solubility but maximum precipitation at the isoelectric point. Non-ionic surfactants were used for size and shape control, and as stabilizing agents. Aerosol performance of the antibody nanoparticles was assessed using Andersen Cascade Impactor. Spinhaler® and Handihaler® were used as model DPI devices. SEM micrographs revealed that the shape of the submicron particles was altered by varying the type of surfactant added to the precipitating medium. Particle size as measured by dynamic light scattering was also varied based on the type and concentration of the surfactant. The surfactants were able to stabilize the IgG during the precipitation process. Polyhedral, sponge-like, and spherical nanoparticles demonstrated improved aerosolization properties compared to irregularly shaped (>20 μm) unprocessed particles. Stable antibody submicron particles of different shapes and sizes were prepared. Careful control of the shape of such particles is critical to ensuring optimized lung delivery by dry powder inhalation.     

Ampicillin Nanoparticles Production via Supercritical CO2 Gas Antisolvent Process

The micronization of ampicillin via supercritical gas antisolvent (GAS) process was studied. The particle size distribution was significantly controlled with effective GAS variables such as initial solute concentration, temperature, pressure, and antisolvent addition rate. The effect of each variable in three levels was investigated. The precipitated particles were analyzed with scanning electron microscopy (SEM) and Zetasizer Nano ZS. The results indicated that decreasing the temperature and initial solute concentration while increasing the antisolvent rate and pressure led to a decrease in ampicillin particle size. The mean particle size of ampicillin was obtained in the range of 220–430 nm by varying the GAS effective variables. The purity of GAS-synthesized ampicillin nanoparticles was analyzed in contrast to unprocessed ampicillin by FTIR and HPLC. The results indicated that the structure of the ampicillin nanoparticles remained unchanged during the GAS process.KEY WORDS: ampicillin, nanoparticles, precipitation, supercritical gas antisolvent     

Preparation,Characterization, and Optimization of Folic Acid-Chitosan-Methotrexate Core-Shell Nanoparticles by Box-Behnken Design for Tumor-Targeted Drug Delivery

The objective of this study was to investigate the combined influence of independent variables in the preparation of folic acid-chitosan-methotrexate nanoparticles (FA-Chi-MTX NPs). These NPs were designed and prepared for targeted drug delivery in tumor. The NPs of each batch were prepared by coaxial electrospray atomization method and evaluated for particle size (PS) and particle size distribution (PSD). The independent variables were selected to be concentration of FA-chitosan, ratio of shell solution flow rate to core solution flow rate, and applied voltage. The process design of experiments (DOE) was obtained with three factors in three levels by Design expert software. Box-Behnken design was used to select 15 batches of experiments randomly. The chemical structure of FA-chitosan was examined by FTIR. The NPs of each batch were collected separately, and morphologies of NPs were investigated by field emission scanning electron microscope (FE-SEM). The captured pictures of all batches were analyzed by ImageJ software. Mean PS and PSD were calculated for each batch. Polynomial equation was produced for each response. The FE-SEM results showed the mean diameter of the core-shell NPs was around 304 nm, and nearly 30% of the produced NPs are in the desirable range. Optimum formulations were selected. The validation of DOE optimization results showed errors around 2.5 and 2.3% for PS and PSD, respectively. Moreover, the feasibility of using prepared NPs to target tumor extracellular pH was shown, as drug release was greater in the pH of endosome (acidic medium). Finally, our results proved that FA-Chi-MTX NPs were active against the human epithelial cervical cancer (HeLa) cells.     

Preparation and evaluation of warfarin-β-cyclodextrin loaded chitosan nanoparticles for transdermal delivery

The main objective of the present work was to prepare warfarin-β-cyclodextrin (WAF-β-CD) loaded chitosan (CS) nanoparticles for transdermal delivery. CS is a hydrophilic carrier therefore, to overcome the hydrophobic nature of WAF and allow its incorporation into CS nanoparticles, WAF was first complexed with β-cyclodextrin (β-CD). CS nanoparticles were prepared by ionotropic pre-gelation using tripolyphosphate (TPP). Morphology, size and structure characterization of nanoparticles were carried out using SEM, TEM and FTIR, respectively. Nanoparticles prepared with 3:1 CS:TPP weight ratio and 2mg/ml final CS concentration were found optimum. They possessed spherical particles (35±12nm diameter) with narrow size distribution (PDI=0.364) and 94% entrapment efficiency. The in vitro release as well as the ex vivo permeation profiles of WAF-β-CD from the selected nanoparticle formulation were studied at different time intervals up to 8h. In vitro release of WAF-β-CD from CS nanoparticles followed a Higuchi release profile whereas its ex vivo permeation (at pH 7.4) followed a zero order permeation profile. Results suggested that the developed WAF-β-CD loaded CS carrier could offer a controlled and constant delivery of WAF transdermally.     

Novel Simvastatin Inhalation Formulation and Characterisation

Simvastatin (SV), a drug of the statin class currently used orally as an anti-cholesterolemic via the inhibition of the 3-hydroxy-3-methyl-glutaryl-Coenzyme A (HMG-CoA) reductase, has been found not only to reduce cholesterol but also to have several other pharmacological actions that might be beneficial in airway inflammatory diseases. Currently, there is no inhalable formulation that could deliver SV to the lungs. In this study, a pressurised metered-dose inhaler (pMDI) solution formulation of SV was manufactured, with ethanol as a co-solvent, and its aerosol performance and physico-chemical properties investigated. A pMDI solution formulation containing SV and 6% w/w ethanol was prepared. This formulation was assessed visually and quantitatively for SV solubility. Furthermore, the aerosol performance (using Andersen Cascade impactor at 28.3 L/min) and active ingredient chemical stability up to 6 months at different storage temperatures, 4 and 25°C, were also evaluated. The physico-chemical properties of the SV solution pMDI were also characterised by differential scanning calorimetry (DSC), thermogravimetric analysis (TGA) and laser diffraction. The aerosol particles, determined using scanning electron microscopy (SEM), presented a smooth surface morphology and were spherical in shape. The aerosol produced had a fine particle fraction of 30.77 ± 2.44% and a particle size distribution suitable for inhalation drug delivery. Furthermore, the short-term chemical stability showed the formulation to be stable at 4°C for up to 6 months, whilst at 25°C, the formulation was stable up to 3 months. In this study, a respirable and stable SV solution pMDI formulation for inhalation has been presented that could potentially be used clinically as an anti-inflammatory therapy for the treatment of several lung diseases.Key Words: lung inflammation, pMDI, pressurised metered dose inhaler, simvastatin     

Calculating expected lung deposition of aerosolized administration of AAV vector in human clinical studies

BACKGROUND: Cystic fibrosis is an autosomal recessive disease affecting approximately 1 in 2500 live births. Introducing the cDNA that codes for normal cystic fibrosis transmembrane conductance regulator (CFTR) to the small airways of the lung could result in restoring the CFTR function. A number of vectors for lung gene therapy have been tried and adeno-associated virus (AAV) vectors offer promise. The vector is delivered to the lung using a breath-actuated jet nebulizer. The purpose of this project was to determine the aerosolized AAV (tgAAVCF) particle size distribution (PSD) in order to calculate target doses for lung delivery. METHODS: A tgAAVCF solution was nebulized using the Pari LC Plus (n = 3), and the PSD was determined by coupling laser diffraction and inertial impaction (NGI) techniques. The NGI allowed for quantification of the tgAAVCF at each stage of impaction, ensuring that rAAV-CFTR vector is present and not empty particles. Applying the results to mathematical algorithms allowed for the calculation of expected pulmonary deposition. RESULTS: The mass median diameter (MMD) for the tgAAVCF was 2.78 +/- 0.43 microm. If the system works ideally and the patient only receives aerosol on inspiration, the patient would receive 47 +/- 0% of the initial dose placed in the nebulizer, with 72 +/- 0.73% of this being deposited beyond the vocal cords. CONCLUSIONS: This technology for categorizing the pulmonary delivery system for lung gene therapy vectors can be adapted for advanced aerosol delivery systems or other vectors.     

Study of the RESS Process for Producing Beclomethasone-17,21-Dipropionate Particles Suitable for Pulmonary Delivery

The purpose of this research was to micronize beclomethasone-17,21-dipropionate (BDP), an anti-inflammatory inhaled corticosteroid commonly used to treat asthma, using the rapid expansion of supercritical solution (RESS) technique. The RESS technique was chosen for its ability to produce both micron particles of high purity for inhalation, and submicron/nano particles as a powder handling aid for use in next generation dry powder inhalers (DPIs). Particle formation experiments were carried out with a capillary RESS system to determine the effect of experimental conditions on the particle size distribution (PSD). The results indicated that the RESS process conditions strongly influenced the particle size and morphology; with the BDP mean particle size decreasing to sub-micron and nanometer dimensions. An increase in the following parameters, i.e. nozzle diameter, BDP mol fraction, system pressure, and system temperature; led to larger particle sizes. Aerodynamic diameters were estimated from the SEM data using three separate relations, which showed that the RESS technique is promising to produce particles suitable for pulmonary delivery.     

SAA-only HDL formed during the acute phase response in apoA-I+/+ and apoA-I-/- mice.

Serum amyloid A (SAA) is an acute phase protein of unknown function that is involved in systemic amyloidosis and may also be involved in atherogenesis. The precise role of SAA in these processes has not been established. SAA circulates in plasma bound to high density lipoprotein-3 (HDL3). The pathway for the production of SAA-containing HDL is not known. To test whether apolipoprotein (apo)A-I-HDL is required in the production of SAA-HDL, we analyzed the lipopolysaccharide (LPS)-induced changes in apoA-I+/+ and apoA-I-/- mice. In apoA-I+/+ mice, after injection of LPS, remodeling of HDL occurred: total cholesterol increased and apoA-I decreased slightly and shifted to lighter density. Dense (density of HDL3) but large (size of HDL2 ) SAA-containing particles were formed. Upon fast phase liquid chromatography fractionation of plasma, >90% of SAA eluted with HDL that was enriched in cholesterol and phospholipid and shifted "leftward" to larger particles. Non-denaturing immunoprecipitation with anti-mouse apoA-I precipitated all of the apoA-I but not all of the SAA, confirming the presence of SAA-HDL devoid of apoA-I. In the apoA-I-/- mice, which normally have very low plasma lipid levels, LPS injection resulted in significantly increased total and HDL cholesterol. Greater than 90% of the SAA was lipid associated and was found on dense but large, spherical HDL particles essentially devoid of other apolipoproteins.We conclude that serum amyloid A (SAA) is able to sequester lipid, forming dense but large HDL particles with or without apoA-I or other apolipoproteins. The capacity to isolate lipoprotein particles containing SAA as the predominant or only apolipoprotein provides an important system to further explore the biological function of SAA.     

Homogeneous Bacterial Aerosols Produced with a Spinning-Disc Generator

Aerosols composed of viable particles of a uniform size were produced with a commercial spinning-disc generator from aqueous suspensions of Bacillus subtilis var. niger spores containing various amounts of an inert material, dextran, to regulate aerosol particle size. Aerosols composed of single naked spores having an equivalent spherical diameter of 0.87 mum were produced from spore suspensions without dextran, whereas aerosols produced from suspensions containing 0.001, 0.01, 0.1, and 1% dextran had median diameters of 0.90, 1.04, 1.80, and 3.62 mum, respectively. Such aerosols, both homogeneous and viable, would be useful for calibrating air sampling devices, evaluating air filter systems, or for employment wherever aerosol behavior may be size-dependent.     

Psychometric and comparative study of an Argentine version of the morningness composite and the early/late preference scales

This study examined the psychometric properties of an Argentine Version of the Morningness Composite (CS) and the Early/Late Preference (PS) Scales. During summer (long photoperiod in Argentina), 304 subjects (69.1% women; mean age=33.64 yrs, SD=14 yrs) completed the scales for the first time. In winter (short photoperiod), 100 of the same individuals (71% women; mean age=37.17 yrs, SD=14.63 yrs) were retested. The total scores ranged within the values reported by previous studies and were independent of gender. Older subjects showed higher morningness scores. The internal consistencies were good (CS=0.86, PS=0.82). Item 7 from the CS and items 7 and 9 from the PS showed low item-scale correlation. Factor analysis produced a three-factor solution for both scales. However, the inconsistency of the evening items suggests that the single-solution may be more acceptable. Test-retest correlations were satisfactory (CS=0.88, PS=0.78), but the two-related-sample test revealed significant differences between test and retest scores, suggesting relative temporal stabilities. Both scales presented similar and acceptable psychometric properties and good correlation, indicating construct validity.