Chemical composition and antioxidant activity of dried powder formulations of Agaricus blazei and Lentinus edodes

Several mushroom species have been pointed out as sources of antioxidant compounds, in addition to their important nutritional value. Agaricus blazei and Lentinus edodes are among the most studied species all over the world, but those studies focused on their fruiting bodies instead of other presentations, such as powdered preparations, used as supplements. In the present work the chemical composition (nutrients and bioactive compounds) and antioxidant activity (free radical scavenging activity, reducing power and lipid peroxidation inhibition) of dried powder formulations of the mentioned mushroom species (APF and LPF, respectively) were evaluated. Powder formulations of both species revealed the presence of essential nutrients, such as proteins, carbohydrates and unsaturated fatty acids. Furthermore, they present a low fat content (<2 g/100 g) and can be used in low-calorie diets, just like the mushrooms fruiting bodies. APF showed higher antioxidant activity and higher content of tocopherols and phenolic compounds (124 and 770 μg/100 g, respectively) than LPF (32 and 690 μg/100 g). Both formulations could be used as antioxidant sources to prevent diseases related to oxidative stress. © 2012 Elsevier Ltd. All rights reserved.

Particle engineering for the production of respirable dry powder formulations

Hyaluronan (HA) plays an important role in lung pathophysiology. For this reason it has attracted great attention both as active ingredient and as excipient in treating lung diseases by direct pulmonary HA administration. The aim was the production of highly respirable and flowable HA powders either as a potential carrier for drug delivery or for being delivered directly by inhalation. Engineered sodium hyaluronate powders were produced by spray-drying technique. All the spray-dried powders were characterised in terms of particle size distribution, drug content, morphology and in vitro respirability. HA was successfully formulated with salbutamol sulphate in combination with leucine and highlighted remarkable aerodynamic performance (emitted dose equal to 83 % and FPF % equal to 97.1%). Moreover, HA colloidal solutions were designed and they were spray-dried. In order to improve particle aerodynamic characteristics, different types of excipients were investigated. In particular, stearylamine (5% w/w) allowed to obtain the best performance throughout the experimental set. Finally, in vitro biocompatibility was carried out by MTT assay and High Content Analysis for selected dry powder formulations and starting materials. The assays demonstrated the same outcome by confirming the HA biocompatibility and by producing the same rank of toxicity for the surfactants. The general conclusion of the project is that formulation containing HA and stearyl alcohol represents the best performing formulation.

Quil A-lipid powder formulations releasing ISCOMs and related colloidal stuctures upon hydration

The aim of the present study was to prepare solid Quil A-cholesterol-phospholid formulations (as powder mixtures or compressed to pellets) by physical mixing or by freeze-drying of aqueous dispersions of these components in ratios that allow spontaneous formation of ISCOMs and other colloidal stuctures upon hydration. The effect of addition of excess cholesterol to the lipid mixtures on the release of a model antigen (PE-FITC-OVA) from the pellets was also investigated. Physical properties were evaluated by X-ray powder diffractometry (XPRD), differential scanning calorimetry (DSC), scanning electron microscopy (SEM), and polarized light microscopy (PLM). Characterization of aqueous colloidal dispersions was performed by negative staining transmission electron microscopy (TEM). Physically mixed powders (with or without PE-FITC-OVA) and pellets prepared from the same powders did not spontaneously form ISCOM matrices and related colloidal structures such as worm-like micelles, ring-like micelles, lipidic/layered structures and lamellae (hexagonal array of ring-like micelles) upon hydration as expected from the pseudo-temary diagram for aqueous mixtures of Quil A, cholesterol and phospholipid. In contrast, spontaneous formation of the expected colloids was demonstrated for the freeze-dried lipid mixtures. Pellets prepared by compression of freeze-dried powders released PE-FITC-OVA slower than those prepared from physically mixed powders. TEM investigations revealed that the antigen was released in the form of colloidal particles (ISCOMs) from pellets prepared by compression of freeze-dried powders. The addition of excess cholesterol slowed down the release of antigen. The findings obtained in this study are important for the formulation of solid Quil A-containing lipid articles as controlled particulate adjuvant containing antigen delivery systems. (c) 2004 Elsevier B.V. All rights reserved.

Chitosan-modified dry powder formulations for pulmonary gene delivery

Spray-drying is an effective process for preparing micron-dimensioned particles for pulmonary delivery. Previously, we have demonstrated enhanced dispersibility and fine particle fraction of spray-dried nonviral gene delivery formulations using amino acids or absorption enhancers as dispersibility-enhancing excipients. In this study, we investigate the use of the cationic polymer chitosan as a readily available and biocompatible dispersibility enhancer. Lactose-lipid:polycation:pDNA (LPD) powders were prepared by spray-drying and post-mixed with chitosan or spray-dried chitosan. In addition, the water-soluble chitosan derivative, trimethyl chitosan, was added to the lactose-LPD formulation before spray-drying. Spray-dried chitosan particles, displaying an irregular surface morphology and diameter of less than 2 mu m, readily adsorbed to lactose-LPD particles following mixing. In contrast with the smooth spherical surface of lactose-LPD particles, spray-dried trimethyl chitosan-lactose-LPD particles demonstrated increased surface roughness and a unimodal particle size distribution (mean diameter 3.4 mu m), compared with the multimodal distribution for unmodified lactose-LPD powders (mean diameter 23.7 mu m). The emitted dose and in vitro deposition of chitosan-modified powders was significantly greater than that of unmodified powders. Moreover, the inclusion of chitosan mediated an enhanced level of reporter gene expression. In summary, chitosan enhances the dispersibility and in vitro pulmonary deposition performance of spray-dried powders.

Effects of magnesium stearate on the efficient dispersion of salbutamol sulphate from carrier-based dry powder inhaler formulations

Dry powder inhaler (DPI) formulations is one of the most useful aerosol preparations in which drugs may be formulated as carrier-based interactive mixtures with micronised drug particles (<5 μm) adhered onto the surface of large inert carriers (lactose powders). The addition of magnesium stearate (MgSt) (1-3), was found to increase dispersion of various drugs from DPI formulations. Recently, some active compounds coated with 5% (wt/wt) MgSt using the mechanofusion method showed significant improvements in aerosolization behavior due to the reduction in intrinsic cohesion force (4). Application of MgSt in powder formulations is not new; however, no studies demonstrated the minimum threshold level for this excipient in efficient aerosolization of drug powders from the interactive mixtures. Therefore, this study investigated the role of MgSt concentration on the efficient dispersion of salbutamol sulphate (SS) from DPI formulations.

A novel cell compatible impingement system to study in vitro drug absorption from dry powder aerosol formulations

A modified Astra type multistage liquid impinger (MSLI) with integrated bronchial cell monolayers was used to study deposition and subsequent drug absorption on in vitro models of the human airway epithelial barrier. Inverted cell culture of Calu-3 cells on the bottom side of cell culture filter inserts was integrated into a compendial MSLI. Upside down cultivation did not impair the barrier function, morphology and viability of Calu-3 cells. Size selective deposition with subsequent absorption was studied for three different commercially available dry powder formulations of salbutamol sulphate and budesonide. After deposition without size separation the absorption rates from the aerosol formulations differed but correlated with the size of the carrier lactose particles. However, after deposition in the MSLI, simulating relevant impaction and causing the separation of small drug crystals from the carrier lactose, the absorption rates of the three formulations were identical, confirming the bioequivalence of the three formulations.

Formulations of Melia azedarach to control diabrotica speciosa (germar) (coleoptera: chrysomelidae) larvae in corn and plant enhancement

We evaluated oil and powder formulations of Melia azedarach for controlling larvae of Diabrotica speciosa (Germar) in corn and plant enhancement. Five concentrations of each formulation were evaluated and compared to fipronil (negative control) and distilled water (positive control). After treatment, the number of surviving insects (larvae, pupae, and adults), the adult body weight, the sex ratio, and the longevity were recorded, while the height, dry weight of aerial part and roots, and number of leaves of plants were measured. The oil formulation at 4.0 mL reduced the larvae population of D. speciosa similarly to the insecticide fipronil, which resulted in greater height, dry weight of the root system, and number of leaves. Powder formulation at concentrations of 40, 80, and 160 mg caused larval mortality above 80%; however, these concentrations did not prevent reduction of plant height and dry weight of aerial part. Further studies assessing the residual period of M. azedarach control against D. speciosa larvae and its phytotoxicity, which are common traits associated with azadirachtin application, are necessary to subsidize the next steps of this alternative control strategy.

Pharmaceutical process optimisation of bulk lyophilisates:implications of powder handling

Lyophilisation or freeze drying is the preferred dehydrating method for pharmaceuticals liable to thermal degradation. Most biologics are unstable in aqueous solution and may use freeze drying to prolong their shelf life. Lyophilisation is however expensive and has seen lots of work aimed at reducing cost. This thesis is motivated by the potential cost savings foreseen with the adoption of a cost efficient bulk drying approach for large and small molecules. Initial studies identified ideal formulations that adapted well to bulk drying and further powder handling requirements downstream in production. Low cost techniques were used to disrupt large dried cakes into powder while the effects of carrier agent concentration were investigated for powder flowability using standard pharmacopoeia methods. This revealed superiority of crystalline mannitol over amorphous sucrose matrices and established that the cohesive and very poor flow nature of freeze dried powders were potential barriers to success. Studies from powder characterisation showed increased powder densification was mainly responsible for significant improvements in flow behaviour and an initial bulking agent concentration of 10-15 %w/v was recommended. Further optimisation studies evaluated the effects of freezing rates and thermal treatment on powder flow behaviour. Slow cooling (0.2 °C/min) with a -25°C annealing hold (2hrs) provided adequate mechanical strength and densification at 0.5-1 M mannitol concentrations. Stable bulk powders require powder transfer into either final vials or intermediate storage closures. The targeted dosing of powder formulations using volumetric and gravimetric powder dispensing systems where evaluated using Immunoglobulin G (IgG), Lactate Dehydrogenase (LDH) and Beta Galactosidase models. Final protein content uniformity in dosed vials was assessed using activity and protein recovery assays to draw conclusions from deviations and pharmacopeia acceptance values. A correlation between very poor flowability (p<0.05), solute concentration, dosing time and accuracy was revealed. LDH and IgG lyophilised in 0.5 M and 1 M mannitol passed Pharmacopeia acceptance values criteria with 0.1-4 while formulations with micro collapse showed the best dose accuracy (0.32-0.4% deviation). Bulk mannitol content above 0.5 M provided no additional benefits to dosing accuracy or content uniformity of dosed units. This study identified considerations which included the type of protein, annealing, cake disruption process, physical form of the phases present, humidity control and recommended gravimetric transfer as optimal for dispensing powder. Dosing lyophilised powders from bulk was demonstrated as practical, time efficient, economical and met regulatory requirements in cases. Finally the use of a new non-destructive technique, X-ray microcomputer tomography (MCT), was explored for cake and particle characterisation. Studies demonstrated good correlation with traditional gas porosimetry (R2 = 0.93) and morphology studies using microscopy. Flow characterisation from sample sizes of less than 1 mL was demonstrated using three dimensional X-ray quantitative image analyses. A platinum-mannitol dispersion model used revealed a relationship between freezing rate, ice nucleation sites and variations in homogeneity within the top to bottom segments of a formulation.

Printability of calcium phosphate: Calcium sulphate powders for the application of tissue engineered bone scaffolds using the 3D printing technique

In this study, calcium phosphate (CaP) powders were blended with a three-dimensional printing (3DP) calcium sulfate (CaSO4)-based powder and the resulting composite powders were printed with a water-based binder using the 3DP technology. Application of a water-based binder ensured the manufacture of CaP:CaSO4 constructs on a reliable and repeatable basis, without long term damage of the printhead. Printability of CaP:CaSO4 powders was quantitatively assessed by investigating the key 3DP process parameters, i.e. in-process powder bed packing, drop penetration behavior and the quality of printed solid constructs. Effects of particle size, CaP:CaSO4 ratio and CaP powder type on the 3DP process were considered. The drop penetration technique was used to reliably identify powder formulations that could be potentially used for the application of tissue engineered bone scaffolds using the 3DP technique. Significant improvements (p < 0.05) in the 3DP process parameters were found for CaP (30-110 μm):CaSO4 powders compared to CaP (< 20 μm):CaSO4 powders. Higher compressive strength was obtained for the powders with the higher CaP:CaSO4 ratio. Hydroxyapatite (HA):CaSO4 powders showed better results than beta-tricalcium phosphate (β-TCP):CaSO4 powders. Solid and porous constructs were manufactured using the 3DP technique from the optimized CaP:CaSO4 powder formulations. High-quality printed constructs were manufactured, which exhibited appropriate green compressive strength and a high level of printing accuracy.

Formulation and delivery of the bacterial antagonist Bacillus subtilis for management of lentil vascular wilt caused by Fusarium oxysporum f. sp lentis

Different formulations of Bacillus subtilis were prepared using standard laboratory protocols. Bacillus subtilis survived in glucose and talc powders at 8.6 and 7.8 log(10) CFU/g, respectively, for 1 year of storage at room temperature compared with 3.5 log(10) CFU/g on a peat formulation. Glasshouse experiments using soil and seed treatments were conducted to test the efficacy of B. subtilis for protecting lentil against the wilt disease caused by Fusariumoxysporum f. sp. lentis. Seed treatments with formulations of B. subtilis on glucose, talc and peat significantly enhanced its biocontrol activity against Fusarium compared with a treatment in which spores were applied directly to seed. The formulations decreased disease severity by reducing colonization of plants by the pathogen, promoting their growth and increased the dry weight of lentil plants. Of these treatments the glucose and talc-based powder formulations were more effective than the peat formulation and the spore application without a carrier. It was shown that the B. subtilis spores applied with glucose were viable for longer than those applied with other carriers. Seed treatment with these formulated spores is an effective delivery system that can provide a conducive environment for B. subtilis to suppress vascular wilt disease on lentil and has the potential for utilization in commercial field application.

Chitosan-based mucoadhesive tablets for oral delivery of ibuprofen

Chitosan and its half-acetylated derivative have been compared as excipients in mucoadhesive tablets containing ibuprofen. Initially the powder formulations containing the polymers and the drug were prepared by either co-spray drying or physical co-grinding. Polymer–drug interactions and the degree of drug crystallinity in these formulations were assessed by infrared spectroscopy and differential scanning calorimetry. Tablets were prepared and their swelling and dissolution properties were studied in media of various pHs. Mucoadhesive properties of ibuprofen-loaded and drug-free tablets were evaluated by analysing their detachment from pig gastric mucosa over a range of pHs. Greater polymer–drug interactions were seen for spray-dried particles compared to co-ground samples and drug loading into chitosan-based microparticles (41%) was greater than the corresponding half-acetylated samples (32%). Swelling and drug release was greater with the half-acetylated chitosan tablets than tablets containing the parent polymer and both tablets were mucoadhesive, the extent of which was dependent on substrate pH. The results illustrate the potential sustained drug delivery benefits of both chitosan and its half-acetylated derivative as mucoadhesive tablet excipients.

Efeitos de genótipos de feijão caupi e de espécies botânicas em diferentes formulações sobre Callosobruchus maculatus (FABR.)

Pós-graduação em Agronomia (Proteção de Plantas) - FCA

Influence of alternative drying aids on water sorption of spray dried mango mix powders: a thermodynamic approach

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Investigating the delivery of antimicrobial proteins and aminoglycoside antibiotics to the airways

Biopharmaceuticals are finding wide applications in the management of diverse disease conditions. Pulmonary delivery of proteins may constitute an effective and efficient non-invasive alternative to parenteral delivery, which is currently the main route of administration of biopharmaceutical drugs. A particular area, in which pulmonary delivery of peptides and proteins may find ready application, is in the local delivery of antimicrobial peptides and proteins to the airway, a measure that could potentially bring about improvements to currently available antipseudomonal therapies. This thesis has therefore sought to develop inhalable antimicrobial proteins in combination with antibiotics that have particularly good antimicrobial activity against Pseudomonas aeruginosa infections in the respiratory tract of people with cystic fibrosis (CF). Through process optimisation, a suitable spray drying method was developed and used for the preparation of active, inhalable dry powder formulations of the antimicrobial protein, lactoferrin, and aminoglycosides (tobramycin and gentamicin). The physicochemical properties, aerosolisation performance and the antibacterial properties of the various spray-dried formulations were assessed. In addition, a relevant in vitro cellular model was employed to investigate the potential cytotoxic and pro-inflammatory effects of the various formulations on four bronchial human epithelial cells together with their effectiveness at reducing bacterial colonies when administered on to biofilm co-cultured on the epithelial cells. It was found that following spray drying the particles obtained were mostly spherical, amorphous and possessed suitable aerosolisation characteristics. The various spray-dried antimicrobial proteins (lactoferrin or apo lactoferrin) and co-spray dried combinations of the proteins and aminoglycosides were found to exhibit bactericidal activity against planktonic and biofilms of P. aeruginosa. In general, the spray drying process was found not to significantly affect the antimicrobial activities of the protein. Treatment of the different bronchial epithelial cell lines with the antimicrobial formulations showed that the various formulations were non-toxic and that the co-spray dried combinations significantly reduced established P. aeruginosa biofilms on the four bronchial epithelial cells. Overall, the results from this thesis demonstrates that spray drying could potentially be employed to prepare inhalable antimicrobial agents comprised of proteins and antibiotics. These new combinations of proteins and aminoglycosides has promising applications in the management of P. aeruginosa in the airway of cystic fibrosis patients.

Studies on the effect of the size of polycaprolactone microspheres for the dispersion of Salbutamol Sulfate from dry powder inhaler formulations

Purpose: To study the effect of the size of the surface-coated polycaprolactone (PCL) microparticle carriers on the aerosolization and dispersion of Salbutamol Sulfate (SS) from Dry Powder Inhaler (DPI) formulations. Methods: The microparticles were fabricated using an emulsion technique in four different sizes (25, 48, 104 and 150 μm) and later coated with Magnesium stearate (MgSt) and leucine. They were characterized by laser diffraction and SEM. The Fine Particle Fraction (FPF) of SS from powder mixtures was determined by a Twin Stage Impinger (TSI). Results: As the carrier size increased from 25 μm to 150 μm, the FPF of the SS delivered by the coated PCL particles increased approximately four fold. A linear relationship was found between the FPF and Volume mean Diameter (VMD) of the particles over this range. Conclusions: The dispersion behaviour of SS from PCL carriers was dependent on the inherent size of the carriers and the increased FPF of SS with increased carrier size probably reflects the higher mechanical forces produced due to the carrier-carrier collisions or collisions between the carrier particles and the internal walls of the inhaler during aerosolization.