919 resultados para Spray deposit
Resumo:
SD Apo Lactoferrin-Tobramycin/Gentamicin Combinations are superior to monotherapy in the eradication of Pseudomonas aeruginosa Biofilm in the lungs Wilson Oguejiofor1, Lindsay J. Marshall1, Andrew J. Ingham1, Robert Price2, Jag. Shur2 1School of Life and Health Sciences, Aston University, Birmingham, UK. 2School of Pharmacy and Pharmacology, University of Bath, Bath, UK. KEYWORDS: lactoferrin, apo lactoferrin, spray drying, biofilm, cystic fibrosis Introduction Chronic lung infections from the opportunistic pathogeen Pseudomonas aeruginosa has been recognised as a major contributor to the incidences of high morbidity and mortality amongst cystic fibrosis (CF) patients (1,2). Currently, strategies for managing lung infections in CF patients involves the aggressive use of aerosolised antibiotics (3), however, increasing evidence suggests that the biofilm component of P. aeruginosa in the lower airway remains unperturbed and is associated with the development of antibiotic resistance. If this is so then, there is an urgent need to suitably adjust the current treatment strategy so that it includes compounds that prevent biofilm formation or disrupt established biofilms. It is well understood that biofilm formation is strongly dependent on iron (Fe3+) availability (4), therefore aerosolised anti-infective formulations which has the ability to chelate iron may essentially be a well suited therapy for eliminating P. aeruginosa biofilms on CF airway epithelial cells (5). In this study, we report the use of combination therapy; an aminoglycosides (tobramycin and gentamicin) and an antimicrobial peptide (lactoferrin) to significantly deplete P. aeruginosa biofilms. We demonstrate that lactoferrin-tobramycin and lactoferrin-gentamicin combinations are superior to the single antibiotic regime currently being employed to combat P. aeruginosa biofilms. MATERIALS AND METHOD Antibiotics: The antibiotics used in this study included gentamicin and tobramycin supplied by Fagron, UK. Bacterial strain and growth conditions: Pseudomonas aeruginosa strain PAO1 was provided by Prof. Peter Lambert of Aston University, Birmingham UK. The Strains were routinely grown from storage in a medium supplemented with magnesium chloride, glucose and casamino acids. Dialysis of lactoferrin: Apo lactoferrin was prepared by dialyzing a suspension of lactoferrin for 24 hrs at 4 °C against 20 mmol/L sodium dihydrogen phosphate, 20 mmol/L sodium acetate and 40 mmol/L EDTA (pH 3.5). Ferric ion (Fe3+) removal was verified by atomic absorption spectroscopy measurements. Spray drying of combinations of lactoferrin and apo lactoferrin with the different aminoglycosides: Combinations of tobramycin and gentamicin with the different preparations of lactoferrin were spray dried (SD) as a 2% (w/v) aqueous suspension. The spray drying parameters utilized for the production of suitable micron-sized particles includes: Inlet temperature, 180°C, spray flow rate, 606 L/hr; pump setting, 10%; aspirator setting, 85% (34m3/hr) to produce various outlet temperatures ranging from 99 - 106°C. Viability assay: To test the bactericidal activity of the various combinations, a viability assay was performed as previously described by Xu, Xiong et al. (6) with some modifications. Briefly, 10µL of ~ c. 6.6 x 107 CFU mL-1 P. aeruginosa strain PAO1 suspension were incubated (37°C, 60 mins) with 90 µL of a 2 µg/mL concentration of the various combinations and sampled every 10 mins. After incubation, the cells were diluted in deionised water and plated in Mueller hinton agar plates. Following 24 h incubation of the plates at 37°C, the percentage of viable cells was determined relative to incubation without added antibiotics. Biofilm assay: To test the susceptibility of the P. aeruginosa strain to various antibiotics in the biofilms mode of growth, overnight cultures of P. aeruginosa were diluted 1:100 into fresh medium supplemented with magnesium chloride, glucose and casamino acids. Aliquots of the dilution were dispensed into a 96 well dish and incubated (37°C, 24 h). Excess broth was removed and the number of colony forming units per milliliter (CFU/mL) of the planktonic bacteria was quantified. The biofilms were then washed and stained with 0.1% (w/v) crystal violet for 15 mins at room temperature. Following vigorous washing with water, the stained biofilms were solubilized in 30% acetic acid and the absorbance at 550nm of a 125 µL aliquot was determined in a microplate reader (Multiskan spectrum, Thermo Scientific) using 30% acetic acid in water as the blank. Aliquots of the broth prior to staining were used as an indicator of the level of planktonic growth. RESULTS AND DISCUSSION Following spray drying, the mean yield, volume weighted mean diameter and moisture content of lactoferrin powder were measured and were as follows (Table 1 and table 2); Table 1: Spray drying parameters FormulationInlet temp (°C)Outlet temp (°C)Airflow rate (L/hr)Mean yield (%)Moisture content (%) SD Lactoferrin18099 - 10060645.2 ±2.75.9 ±0.4 SD Apo Lactoferrin180100 - 10260657.8 ±1.85.7 ±0.2 Tobramycin180102 - 10460682.1 ±2.23.2 ±0.4 Lactoferrin + Tobramycin180104 - 10660687.5 ±1.43.7 ±0.2 Apo Lactoferrin + Tobramycin180103 - 10460676.3 ±2.43.3 ±0.5 Gentamicin18099 - 10260685.4 ±1.34.0 ±0.2 Lactoferrin + Gentamicin180102 - 10460687.3 ±2.13.9 ±0.3 Apo Lactoferrin + Gentamicin18099 -10360680.1±1.93.4 ±0.4 Table 2: Particle size distribution d10 d50d90 SD Lactoferrin1.384.9111.08 SD Apo Lactoferrin1.284.7911.04 SD Tobramycin1.254.9011.29 SD Lactoferrin + Tobramycin1.175.2715.23 SD Apo Lactoferrin + Tobramycin1.115.0614.31 SD Gentamicin1.406.0614.38 SD Lactoferrin + Gentamicin1.476.2314.41 SD Apo Lactoferrin + Gentamicin1.465.1511.53 The bactericidal activity of the various combinations were tested against P. aeruginosa PAO1 following a 60 minute incubation period (Figure 1 and Figure 2). While 2 µg/mL of a 1:1 combination of spray dried apo lactoferrin and Gentamicin was able to completely kill all bacterial cells within 40 mins, the same concentration was not as effective for the other antibiotic combinations. However, there was an overall reduction of bacterial cells by over 3 log units by the other combinations within 60 mins. Figure 1: Logarithmic plot of bacterial cell viability of various combinations of tobramycin and lactoferrin preparations at 2µg/mL (n = 3). Figure 2: Logarithmic plot of bacterial cell viability of various combinations of gentamicin and lactoferrin preparations at 2µg/mL (n = 3). Crystal violet staining showed that biofilm formation by P. aeruginosa PAO1 was significantly (ANOVA, p < 0.05) inhibited in the presence of the different lactoferrin preparations. Interestingly, apo lactoferrin and spray dried lactoferrin exhibited greater inhibition of both biofilm formation and biofilm persistence (Figure 2). Figure 2: Crystal violet staining of residual biofilms of P. aeruginosa following a 24hr incubation with the various combinations of antibiotics and an exposure to 48 hr formed biofilms. CONCLUSION In conclusion, combination therapy comprising of an antimicrobial peptide (lactoferrin) and an aminoglycosides (tobramycin or gentamicin) provides a feasible and alternative approach to monotherapy since the various combinations are more efficient than the respective monotherapy in the eradication of both planktonic and biofilms of P. aeruginosa. ACKNOWLEDGEMENT The authors would like to thank Mr. John Swarbrick and Friesland Campina for their generous donation of the Lactoferrin. REFERENCES 1.Hassett, D.J., Sutton, M.D., Schurr, M.J., Herr, A.B., Caldwell, C.C. and Matu, J.O. (2009), "Pseudomonas aeruginosa hypoxic or anaerobic biofilm infections within cystic fibrosis airways". Trends in Microbiology, 17, 130-138. 2.Trust, C.F. (2009), "Antibiotic treatment for cystic fibrosis". Report of the UK Cystic Fibrosis Trust Antibiotic Working Group. Consensus document. London: Cystic Fibrosis Trust. 3.Garcia-Contreras, L. and Hickey, A.J. (2002), "Pharmaceutical and biotechnological aerosols for cystic fibrosis therapy". Advanced Drug Delivery Reviews, 54, 1491-1504. 4.O'May, C.Y., Sanderson, K., Roddam, L.F., Kirov, S.M. and Reid, D.W. (2009), "Iron-binding compounds impair Pseudomonas aeruginosa biofilm formation, especially under anaerobic conditions". J Med Microbiol, 58, 765-773. 5.Reid, D.W., Carroll, V., O'May, C., Champion, A. and Kirov, S.M. (2007), "Increased airway iron as a potential factor in the persistence of Pseudomonas aeruginosa infection in cystic fibrosis". European Respiratory Journal, 30, 286-292. 6.Xu, G., Xiong, W., Hu, Q., Zuo, P., Shao, B., Lan, F., Lu, X., Xu, Y. and Xiong, S. (2010), "Lactoferrin-derived peptides and Lactoferricin chimera inhibit virulence factor production and biofilm formation in Pseudomonas aeruginosa". J Appl Microbiol, 109, 1311-1318.
Resumo:
The size frequency distributions of diffuse, primitive and classic beta/A4 deposits was studied in single sections in the hippocampus, parahippocampal gyrus (PHG) and lateral occipitotemporal gyrus (LOT) in five cases of Alzheimer's disease. In most brain regions, the size distribution of the diffuse deposits was significantly different from that of the primitive and classic deposits. The data suggested that larger diffuse deposits appeared to be converted less often into primitive and classic deposits. Significant differences in the size distribution of primitive deposits were commonly observed between brain regions in which there was no difference in the size distribution of the diffuse deposits. Hence, local brain factors may influence the size of diffuse deposit which can be converted into mature amyloid deposit.
Resumo:
The spatial patterns of diffuse, primitive, classic (cored) and compact (burnt-out) subtypes of beta/A4 deposits were studied in coronal sections of the frontal lobe and hippocampus, including the adjacent gyri, in nine cases of Alzheimer's disease (AD). If the more mature deposits were derived from the diffuse deposits then there should be a close association between their spatial patterns in a brain region. In the majority of tissues examined, all deposit subtypes occurred in clusters which varied in dimension from 200 to 6400 microns. In many tissues, the clusters appeared to be regularly spaced parallel to the pia or alveus. The mean dimension of the primitive deposit clusters was greater than those of the diffuse, classic and compact types. In about 60% of cortical tissues examined, the clusters of primitive and diffuse deposits were not in phase, i.e. they alternated along the cortical strip. Clusters of classic deposits appeared to be distributed independently of the diffuse deposit clusters. Cluster size of the primitive deposits was positively correlated with the density of the primitive deposits in a tissue but no such relationship could be detected for the diffuse deposits. This study suggested that there was a complex relationship between the clusters of the different subtypes of beta/A4 deposits. If the diffuse deposits do give rise to the primitive and classic varieties then factors unrelated to the initial deposition of beta/A4 in the form of diffuse plaques were important in the formation of the mature deposits.
Resumo:
Spray drying is widely used to manufacture many powdered products, with the drying process parameters having significant influence over the final powder's surface properties and propensity for unwanted caking. In most cases caking experiments are performed on bulk powders, but especially in multi-component powders, it is often difficult to interpret these results, where interaction effects between particles can be complex. Here the technique of scanning probe microscopy is used to characterize the nanoscale properties of spray dried model milk powders in order to investigate the surface properties of the powders.
Resumo:
The surface composition of food powders created from spray drying solutions containing various ratios of sodium caseinate, maltodextrin and soya oil have been analysed by Electron Spectroscopy for Chemical Analysis. The results show significant enrichment of oil at the surface of particles compared to the bulk phase, and (when the non-oil components only are considered), a significant surface enrichment of sodium caseinate also. The study found evidence of high levels (80%) of surface fat even on particles of food industry grade (92.5%) sodium caseinate containing only 1% fat.
Resumo:
Moisture migration caking of pharmaceutical excipients in the absence of load is a significant quality and stability issue. This study uses Atomic Force Microscopy (AFM) to examine a solid bridge formed between two 20µm spray-dried sodium carbonate particles. The bridge is grown by repeatedly exposing the system to 70% RH and 30% RH cycles at 25?C. A comparison is made with the idealised bridge model developed by Tanaka (1978) which was previously verified using crystalline systems. The resulting system was found to be more complex and grew in two stages. The first stage consisted of linear growth to 5 cycles, followed by a more gradual expansion and the appearance of crystalline structures.
Resumo:
Background: Pulmonary gene therapy requires aerosolisation of the gene vectors to the target region of the lower respiratory tract. Pulmonary absorption enhancers have been shown to improve the penetration of pharmaceutically active ingredients in the airway. In this study, we investigate whether certain absorption enhancers may also enhance the aerosolisation properties of spray-dried powders containing non-viral gene vectors. Methods: Spray-drying was used to prepare potentially respirable trehalose-based dry powders containing lipid-polycation-pDNA (LPD) vectors and absorption enhancers. Powder morphology and particle size were characterised using scanning electron microscopy and laser diffraction, respectively, with gel electrophoresis used to assess the structural integrity of the pDNA. The biological functionality of the powders was quantified using in vitro cell (A549) transfection. Aerosolisation from a Spinhaler® dry powder inhaler into a multistage liquid impinger (MSLI) was used to assess the in vitro dispersibility and deposition of the powders. Results: Spray-dried powder containing dimethyl-β-cyclodextrin (DMC) demonstrated substantially altered particle morphology and an optimal particle size distribution for pulmonary delivery. The inclusion of DMC did not adversely affect the structural integrity of the LPD complex and the powder displayed significantly greater transfection efficiency as compared to unmodified powder. All absorption enhancers proffered enhanced powder deposition characteristics, with the DMC-modified powder facilitating high deposition in the lower stages of the MSLI. Conclusions: Incorporation of absorption enhancers into non-viral gene therapy formulations prior to spray-drying can significantly enhance the aerosolisation properties of the resultant powder and increase biological functionality at the site of deposition in an in vitro model. Copyright © 2005 John Wiley & Sons, Ltd.
Resumo:
This paper compares the crack growth resistance of an experimental spray-formed extrusion with that of a commercial aluminium alloy, the two alloys having similar compositions but markedly different grain structures. Tensile and fracture behaviour is similar in both materials and is influenced by inclusion content. The two materials differ in their crack growth resistance, which is shown to be dependent upon grain size and shape. Environmentally-induced crack growth is favoured by aligned grain boundaries and small grain size.
Resumo:
Spray-drying represents a viable alternative to freeze-drying for preparing dry powder dispersions for delivering macromolecules to the lung. The dispersibility of spray-dried powders is limited however, and needs to be enhanced to improve lung deposition and subsequent biological activity. In this study, we investigate the utility of leucine as a dry powder dispersibility enhancer when added prior to spray-drying a model non-viral gene therapy formulation (lipid:polycation:pDNA, LPD). Freeze-dried lactose-LPD, spray-dried lactose-LPD and spray-dried leucine-lactose-LPD powders were prepared. Scanning electron microscopy showed that leucine, increased the surface roughness of spray-dried lactose particles. Particle size analysis revealed that leucine-containing spray-dried powders were unimodally dispersed with a mean particle diameter of 3.12 μm. Both gel electrophoresis and in vitro cell (A549) transfection showed that leucine may compromise the integrity and biological functionality of the gene therapy vector. The deposition of the leucine containing powder was however significantly enhanced as evidenced by an increase in gene expression mediated by dry powder collected at lower stages of a multistage liquid impinger (MSLI). Further studies are required to determine the potential of leucine as a ubiquitous dispersibility enhancer for a variety of pulmonary formulations. © 2003 Taylor & Francis Ltd.
Resumo:
Abstract The surface compositions of food powders created from spray drying solutions containing various ratios of sodium caseinate, maltodextrin and soya oil have been analysed by Electron Spectroscopy for Chemical Analysis. The results show significant enrichment of oil at the surface of particles compared to the bulk phase and, when the non-oil components only are considered, a significant surface enrichment of sodium caseinate also. The degree of surface enrichment of both oil and sodium caseinate was found to increase with decreasing bulk levels of the respective components. Surface enrichment of oil was also affected by processing conditions (emulsion drop size and drying temperature), but surface enrichment of sodium caseinate was relatively insensitive to these. The presence of "pock marks" on the particle surfaces strongly suggests that the surface oil was caused by rupturing of emulsion droplets at the surface as the surrounding matrix contracts and hardens. © 2013 Elsevier Ltd. All rights reserved.
Resumo:
Purpose: The aim of this work was to optimize biodegradable polyester poly(glycerol adipate-co-ω-pentadecalactone), PGA-co-PDL, microparticles as sustained release (SR) carriers for pulmonary drug delivery. Methods: Microparticles were produced by spray drying directly from double emulsion with and without dispersibility enhancers (L-arginine and L-leucine) (0.5-1.5%w/w) using sodium fluorescein (SF) as a model hydrophilic drug. Results: Spray-dried microparticles without dispersibility enhancers exhibited aggregated powders leading to low fine particle fraction (%FPF) (28.79±3.24), fine particle dose (FPD) (14.42±1.57 μg), with a mass median aerodynamic diameter (MMAD) 2.86±0.24 μm. However, L-leucine was significantly superior in enhancing the aerosolization performance ( L-arginine:%FPF 27.61±4.49-26.57±1.85; FPD 12.40±0.99-19.54±0.16 μg and MMAD 2.18±0.35-2. 98±0.25 μm, L-leucine:%FPF 36.90±3.6-43.38±5. 6; FPD 18.66±2.90-21.58±2.46 μg and MMAD 2.55±0.03-3. 68±0.12 μm). Incorporating L-leucine (1.5%w/w) reduced the burst release (24.04±3.87%) of SF compared to unmodified formulations (41.87±2.46%), with both undergoing a square root of time (Higuchi's pattern) dependent release. Comparing the toxicity profiles of PGA-co-PDL with L-leucine (1.5%w/w) (5 mg/ml) and poly(lactide-co-glycolide), (5 mg/ml) spray-dried microparticles in human bronchial epithelial 16HBE14o-cell lines, resulted in cell viability of 85.57±5.44 and 60.66±6.75%, respectively, after 72 h treatment. Conclusion:The above data suggest that PGA-co-PDL may be a useful polymer for preparing SR microparticle carriers, together with dispersibility enhancers, for pulmonary delivery. © Springer Science+Business Media, LLC 2011.
Resumo:
The use of antimicrobial peptides and proteins as potential therapeutic agents in the management of multi-drug resistant infections is considered an attractive concept especially since such compounds should theoretically have low immunogenicity, high bioavailability with negligible toxicity. In this study we investigated the potential of developing a dry powder inhaler formulation of lactoferrin (a multifunctional iron binding protein). To achieve this, the protein was spray dried from a water only feedstock with suitably adjusted spray drying parameters. The particle size, degree of crystallinity, moisture content and yield of the spray dried powders along with the minimum bactericidal concentration (MBC) against Pseudomonas aeruginosa strain PAO1, were assessed. Dry powder inhaler formulations were prepared, and in vitro assessment studies using the multistage impinger were carried out to assess the aerosolisation performance of the formulations. Data obtained indicate that spray dried lactoferrin retains activity against biofilms and may be successfully employed in the treatment of chronic airway infections.
Resumo:
The spray zone is an important region to control nucleation of granules in a high shear granulator. In this study, a spray zone with cross flow is quantified as a well-mixed compartment in a high shear granulator. Granulation kinetics is quantitatively derived at both particle-scale and spray zone-scale. Two spatial decay rates, DGSDR (droplet-granule spatial decay rate) ζDG and DPSDR (droplet-primary particle spatial decay rate) ζDP, which are functions of volume fraction and diameter of particulate species within the powder bed, are defined to simplify the deduction. It is concluded that in cross flow, explicit analytical results show that the droplet concentration is subject to exponential decay with depth which produces a numerically infinite depth of spray zone in a real penetration process. In a well-mixed spray zone, the depth of the spray zone is 4/(ζDG + ζDP) and π2/3(ζDG + ζDP) in cuboid and cylinder shape, respectively. The first-order droplet-based collision rates of, nucleation rate B0 and rewetting rate RW0 are uncorrelated with the flow pattern and shape of the spray zone. The second-order droplet-based collision rate, nucleated granule-granule collision rate RGG, is correlated with the mixing pattern. Finally, a real formulation case of a high shear granulation process is used to estimate the size of the spray zone. The results show that the spray zone is a thin layer at the powder bed surface. We present, for the first time, the spray zone as a well-mixed compartment. The granulation kinetics of a well-mixed spray zone could be integrated into a Population Balance Model (PBM), particularly to aid development of a distributed model for product quality prediction.
Resumo:
The use of sodium carboxymethylcellulose (NaCMC) as a spray-drying excipient in the preparation of inhalable formulations of proteins was investigated, using alkaline phosphatase as a model functional protein. Two spray-dried powders were investigated: a control powder comprising 100% (w/w) alkaline phosphatase and a test powder comprising 67% (w/w) NaCMC and 33% (w/w) alkaline phosphatase. Following physicochemical characterisation, the powders were prepared as both dry powder inhaler (DPI) and pressurised metered dose inhaler (pMDI) formulations. The aerosolisation performance of the formulations was assessed using a Multi-Stage Liquid Impinger, both immediately after preparation and over a 16-week storage period. Formulating the control powder as a DPI resulted in a poor fine particle fraction (FPF: 10%), whereas the FPF of the NaCMC-modified DPI formulation was significantly greater (47%). When the powders were formulated as pMDI systems, the control and NaCMC-modified powders demonstrated FPFs of 52% and 55%, respectively. Following storage, reduced FPF was observed for all formulations except the NaCMC-modified pMDI system; the performance of this formulation following storage was statistically equivalent to that immediately following preparation. Co-spray-drying proteins and peptides with NaCMC may therefore offer an alternative method for the preparation of stable and respirable pMDI formulations for pulmonary delivery. © 2010 Elsevier B.V.
Resumo:
The present research concentrates on the fabrication of bulk aluminum matrix nanocomposite structures with carbon nanotube reinforcement. The objective of the work was to fabricate and characterize multi-walled carbon nanotube (MWCNT) reinforced hypereutectic Al-Si (23 wt% Si, 2 wt% Ni, 1 wt% Cu, rest Al) nanocomposite bulk structure with nanocrystalline matrix through thermal spray forming techniques viz. plasma spray forming (PSF) and high velocity oxy-fuel (HVOF) spray forming. This is the first research study, which has shown that thermal spray forming can be successfully used to synthesize carbon nanotube reinforced nanocomposites. Microstructural characterization based on quantitative microscopy, scanning and transmission electron microscopy (SEM and TEM), energy dispersive spectroscopy (EDS), X-ray diffraction (XRD), Raman spectroscopy and X ray photoelectron spectroscopy (XPS) confirms (i) retention and macro/sub-macro level homogenous distribution of multiwalled carbon nanotubes in the Al-Si matrix and (ii) evolution of nanostructured grains in the matrix. Formation of ultrathin β-SiC layer on MWCNT surface, due to chemical reaction of Si atoms diffusing from Al-Si alloy and C atoms from the outer walls of MWCNTs has been confirmed theoretically and experimentally. The presence of SiC layer at the interface improves the wettability and the interfacial adhesion between the MWCNT reinforcement and the Al-Si matrix. Sintering of the as-sprayed nanocomposites was carried out in an inert environment for further densification. As-sprayed PSF nanocomposite showed lower microhardness compared to HVOF, due to the higher porosity content and lower residual stress. The hardness of the nanocomposites increased with sintering time due to effective pore removal. Uniaxial tensile test on CNT-bulk nanocomposite was carried out, which is the first ever study of such nature. The tensile test results showed inconsistency in the data attributed to inhomogeneous microstructure and limitation of the test samples geometry. The elastic moduli of nanocomposites were computed using different micromechanics models and compared with experimentally measured values. The elastic moduli of nanocomposites measured by nanoindentation technique, increased gradually with sintering attributed to porosity removal. The experimentally measured values conformed better with theoretically predicted values, particularly in the case of Hashin-Shtrikman bound method.