Investigation of the enhanced antimicrobial activity of combination dry powder inhaler formulations of lactoferrin

The airways of most people with cystic fibrosis are colonized with biofilms of the Gram-negative, opportunistic pathogen Pseudomonas aeruginosa. Delivery of antibiotics directly to the lung in the form of dry powder aerosols offers the potential to achieve high local concentrations directly to the biofilms. Unfortunately, current aerosolised antibiotic regimes are unable to efficiently eradicate these biofilms from the airways. We investigated the ability of the innate antimicrobial, lactoferrin, to enhance the activity of two aminoglycoside antibiotics (tobramycin and gentamicin) against biofilms of P. aeruginosa strain PAO1. Biofilms were prepared in 96 well polystyrene plates. Combinations of the antibiotics and various lactoferrin preparations were spray dried. The bacterial cell viability of the various spray dried combinations was determined. Iron-free lactoferrin (apo lactoferrin) induced a 3-log reduction in the killing of planktonic cell by the aminoglycoside antibiotics (p < 0.01) and also reduced both the formation and persistence of P. aeruginosa biofilms (p < 0.01). Combinations of lactoferrin and an aminoglycoside displays potential as an effective new therapeutic strategy in the treatment of P. aeruginosa biofilms infections such as those typical of the CF lungs.

Characterisation and surface-profiling techniques for composite particles produced by dry powder coating in pharmaceutical drug delivery

The production of composite particles using dry powder coating is a one-step, environmentally friendly, process for the fabrication of particles with targeted properties and favourable functionalities. Diverse functionalities, such flowability enhancement, content uniformity, and dissolution, can be developed from dry particle coating. In this review, we discuss the particle functionalities that can be tailored and the selection of characterisation techniques relevant to understanding their molecular basis. We address key features in the powder blend sampling process and explore the relevant characterisation techniques, focussing on the functionality delivered by dry coating and on surface profiling that explores the dynamics and surface characteristics of the composite blends. Dry particle coating is a solvent- and heat-free process that can be used to develop functionalised particles. However, assessment of the resultant functionality requires careful selection of sensitive analytical techniques that can distinguish particle surface changes within nano and/or micrometre ranges.

Dry powder inhalation of macromolecules using novel PEG-co-polyester microparticle carriers

This study investigated optimizing the formulation parameters for encapsulation of a model mucinolytic enzyme, a-chymotrypsin (a-CH), within a novel polymer; poly(ethylene glycol)-co-poly(glycerol adipate-co-?-pentadecalactone), PEG-co-(PGA-co-PDL) which were then applied to the formulation of DNase I. a-CH or DNase I loaded microparticles were prepared via spray drying from double emulsion (w(1)/o/w(2)) utilizing chloroform (CHF) as the organic solvent, l-leucine as a dispersibility enhancer and an internal aqueous phase (w(1)) containing PEG4500 or Pluronic(®) F-68 (PLF68). a-CH released from microparticles was investigated for bioactivity using the azocasein assay and the mucinolytic activity was assessed utilizing the degradation of mucin suspension assay. The chemical structure of PEG-co-(PGA-co-PDL) was characterized by (1)H NMR and FT-IR with both analyses confirming PEG incorporated into the polymer backbone, and any unreacted units removed. Optimum formulation a-CH-CHF/PLF68, 1% produced the highest bioactivity, enzyme encapsulation (20.08±3.91%), loading (22.31±4.34µg/mg), FPF (fine particle fraction) (37.63±0.97%); FPD (fine particle dose) (179.88±9.43µg), MMAD (mass median aerodynamic diameter) (2.95±1.61µm), and the mucinolytic activity was equal to the native non-encapsulated enzyme up to 5h. DNase I-CHF/PLF68, 1% resulted in enzyme encapsulation (17.44±3.11%), loading (19.31±3.27µg/mg) and activity (81.9±2.7%). The results indicate PEG-co-(PGA-co-PDL) can be considered as a potential biodegradable polymer carrier for dry powder inhalation of macromolecules for treatment of local pulmonary diseases.

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.

Functionalised particles using dry powder coating in pharmaceutical drug delivery:promises and challenges

Introduction: Production of functionalised particles using dry powder coating is a one-step, environmentally friendly process that paves the way for the development of particles with targeted properties and diverse functionalities. Areas covered: Applying the first principles in physical science for powders, fine guest particles can be homogeneously dispersed over the surface of larger host particles to develop functionalised particles. Multiple functionalities can be modified including: flowability, dispersibility, fluidisation, homogeneity, content uniformity and dissolution profile. The current publication seeks to understand the fundamental underpinning principles and science governing dry coating process, evaluate key technologies developed to produce functionalised particles along with outlining their advantages, limitations and applications and discusses in detail the resultant functionalities and their applications. Expert opinion: Dry particle coating is a promising solvent-free manufacturing technology to produce particles with targeted functionalities. Progress within this area requires the development of continuous processing devices that can overcome challenges encountered with current technologies such as heat generation and particle attrition. Growth within this field requires extensive research to further understand the impact of process design and material properties on resultant functionalities.

Modified release of beclometasone dipropionate from chitosan-basedd spray-dried respirable powders

Dry powders for inhalation were prepared by spray drying a 30% v/v aqueous ethanol formulation containing beclometasone dipropionate (BDP), lactose, leucine and chitosan (low, medium or high molecular weight (MW), or combinations thereof). Following physical characterisation of the powders, the aerosolisation and dissolution properties of the powders were investigated using Multi-Stage Liquid Impinger and USP II dissolution apparatus, respectively. The powders were highly dispersible, with emitted doses in excess of 90% of loaded powder aerosolised from a Spinhaler dry powder inhaler. The fine particle fraction (FPF) was observed to decrease, whereas the time for 100% drug release increased, with increasing chitosan MW. For example, the low MW formulation exhibited an FPF of 64% and a 100% dissolution time of 2 h, whereas the high MW formulation demonstrated an FPF of 54% and a dissolution time of 12 h. These powders would be anticipated to deposit predominately in the lower regions of the lung following inhalation, and then undergo delayed rather than instantaneous drug release, offering the potential to reduce dosing frequency and improve patient compliance. (c) 2008 Elsevier B.V. All rights reserved.

The use of absorption enhancers to enhance the despersibility of spray-dried powders for pulmonary gene therapy

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.

Spray drying lactoferrin produces inhalable antimicrobial particles

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.

The use of sodium carboxymethylcellulose in the preparation of spray-dried proteins for pulmonary drug delivery

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.

Poly (D,L-Lactide) based microspheres for pulmonary delivery of proteins

Initial work focused on the preparation, optimisation and characterisation of poly (D,L-lactide) (PLA) microspheres with the aim of optimising their formulation based on minimizing the particle size into the range suitable for pulmonary delivery to alveoli. In order to produce dry powders and to enhance their long-term physico-chemical stability, microspheres were prepared as a dry powder via freeze-drying. Optimisation studies showed that using appropriate concentrations of polymer 3% (w/v) in organic phase and emulsifier 10% (w/v) in external aqueous phase, the double solvent evaporation method produced high protein loading microspheres (72 ± 0.5%) with an appropriate particle size for pulmonary drug delivery. Combined use of trehalose and leucine as cyroprotectants (6% and 1% respectively, w/v) produced freeze-dried powders with the best aerosolisation profile among those tested. Although the freeze-dried PLA microsphere powders were not particularly respirable in dry powder inhalation, nebulisation of the rehydrated powders using an ultrasonic nebuliser resulted in improved aerosilisation performance compared to the air-jet nebuliser. When tested in vitro using a macrophage cell line, the PLA microspheres system exhibited a low cytotoxicity and the microspheres induced phagocytic activity in macrophages. However, interestingly, the addition of an immunomodulator to the microsphere formulations (4%, w/w of polymer) reduced this phagocytic activity and macrophage activation compared to microspheres formulated using PLA alone. This suggested that the addition of trehalose dibehenate may not enhance the ability of these microspheres to be used as vaccine delivery systems.

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.

Enhanced dispersibility and deposition of spray-dried powders for pulmonary gene therapy

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.

Microparticle surface layering through dry coating:impact of moisture content and process parameters on the properties of orally disintegrating tablets

OBJECTIVES: The aim of this study was to investigate the influence of process parameters during dry coating on particle and dosage form properties upon varying the surface adsorbed moisture of microcrystalline cellulose (MCC), a model filler/binder for orally disintegrating tablets (ODTs). METHODS: The moisture content of MCC was optimised using the spray water method and analysed using thermogravimetric analysis. Microproperty/macroproperty assessment was investigated using atomic force microscopy, nano-indentation, scanning electron microscopy, tablet hardness and disintegration testing. KEY FINDINGS: The results showed that MCC demonstrated its best flowability at a moisture content of 11.2% w/w when compared to control, comprising of 3.9% w/w moisture. The use of the composite powder coating process (without air) resulted in up to 80% increase in tablet hardness, when compared to the control. The study also demonstrated that surface adsorbed moisture can be displaced upon addition of excipients during dry processing circumventing the need for particle drying before tabletting. CONCLUSIONS: It was concluded that MCC with a moisture content of 11% w/w provides a good balance between powder flowability and favourable ODT characteristics.