Nebulised lipid-polymer hybrid nanoparticles for the delivery of a therapeutic anti-inflammatory microRNA to bronchial epithelial cells.

Modulation of microRNAs (miRNAs), endogenous regulators of gene expression, is a promising strategy for tackling inflammatory lung diseases. In this proof-of-concept study, we tested delivery of miR-17 to bronchial epithelial cells (BECs) using nebulised lipid-polymer hybrid nanoparticles (LPNs). The primary aim was to reduce the induced secretion of miR-17's target, i.e. the pro-inflammatory chemokine interleukin (IL)-8. Synthetic miR-17 mimics were loaded into LPNs composed of poly(dl-lactic-co-glycolic acid) (PLGA) and the cationic lipid 1,2-dioleoyloxy-3-(trimethylammonium)propane (DOTAP) using a double emulsion solvent evaporation method and nebulised using the Aerogen Solo nebuliser. The physicochemical, aerosol, inflammatory and cytotoxic properties of LPNs were characterised. The effect of LPNs on lipopolysaccharide (LPS)-induced IL-8 production from human NuLi-1 BECs was tested by ELISA. The z-average, polydispersity index and ζ-potential of the LPNs and the aerodynamic properties of nebulised suspensions were in a range optimal for deposition in the bronchi and bronchioles post-inhalation. Cytotoxic and pro-inflammatory effects were minimal for LPNs loaded with a model cargo. Nebulisation did not affect the physicochemical or functional properties of the LPNs. Nebulised miR-17-loaded LPNs downregulated LPS-induced IL-8 secretion by >40% in BECs. This study suggests that DOTAP-modified PLGA LPNs are efficient and well-tolerated carriers for delivery of miRNA mimics to BECs.

Modified Vaccinia Virus Ankara Preferentially Targets Antigen Presenting Cells In Vitro, Ex Vivo and In Vivo.

Modified Vaccinia virus Ankara (MVA) is a promising vaccine vector with an excellent safety profile. However, despite extensive pre-clinical and clinical testing, surprisingly little is known about the cellular tropism of MVA, especially in relevant animal species. Here, we performed in vitro, ex vivo and in vivo experiments with recombinant MVA expressing green fluorescent protein (rMVA-GFP). In both human peripheral blood mononuclear cells and mouse lung explants, rMVA-GFP predominantly infected antigen presenting cells. Subsequent in vivo experiments performed in mice, ferrets and non-human primates indicated that preferential targeting of dendritic cells and alveolar macrophages was observed after respiratory administration, although subtle differences were observed between the respective animal species. Following intramuscular injection, rMVA-GFP was detected in interdigitating cells between myocytes, but also in myocytes themselves. These data are important in advancing our understanding of the basis for the immunogenicity of MVA-based vaccines and aid rational vaccine design and delivery strategies.

Optimization and Dose Estimation of Aerosol Delivery to Non-Human Primates.

BACKGROUND: In pre-clinical animal studies, the uniformity of dosing across subjects and routes of administration is a crucial requirement. In preparation for a study in which aerosolized live-attenuated measles virus vaccine was administered to cynomolgus monkeys (Macaca fascicularis) by inhalation, we assessed the percentage of a nebulized dose inhaled under varying conditions. METHODS: Drug delivery varies with breathing parameters. Therefore we determined macaque breathing patterns (tidal volume, breathing frequency, and inspiratory to expiratory (I:E) ratio) across a range of 3.3-6.5 kg body weight, using a pediatric pneumotachometer interfaced either with an endotracheal tube or a facemask. Subsequently, these breathing patterns were reproduced using a breathing simulator attached to a filter to collect the inhaled dose. Albuterol was nebulized using a vibrating mesh nebulizer and the percentage inhaled dose was determined by extraction of drug from the filter and subsequent quantification. RESULTS: Tidal volumes ranged from 24 to 46 mL, breathing frequencies from 19 to 31 breaths per minute and I:E ratios from 0.7 to 1.6. A small pediatric resuscitation mask was identified as the best fitting interface between animal and pneumotachometer. The average efficiency of inhaled dose delivery was 32.1% (standard deviation 7.5, range 24%-48%), with variation in tidal volumes as the most important determinant. CONCLUSIONS: Studies in non-human primates aimed at comparing aerosol delivery with other routes of administration should take both the inter-subject variation and relatively low efficiency of delivery to these low body weight mammals into account.

Aerosol-mediated delivery of AAV2/6-IκBα attenuates lipopolysaccharide-induced acute lung injury in rats.

Inhibition of the proinflammatory transcription factor NF-κB has previously been shown to attenuate the inflammatory response in tissue after injury. However, the feasibility and efficacy of aerosolized adeno-associated viral (AAV) vector-delivered transgenes to inhibit the NF-κB pathway are less clear. Initial studies optimized the AAV vector for delivery of transgenes to the pulmonary epithelium. The effect of repeated nebulization on the integrity and transduction efficacy of the AAV vector was then examined. Subsequent in vivo studies examined the efficacy of aerosolized rAAV2/6 overexpressing the NF-κB inhibitor IκBα in a rodent endotoxin-induced lung injury model. Initial in vitro investigations indicated that rAAV2/6 was the most effective vector to transduce the lung epithelium, and maintained its integrity and transduction efficacy after repeated nebulization. In our in vivo studies, animals that received aerosolized rAAV2/6-IκBα demonstrated a significant increase in total IκBα levels in lung tissue relative to null vector-treated animals. Aerosolized rAAV2/6-IκBα attenuated endotoxin-induced bronchoalveolar lavage-detected neutrophilia, interleukin-6 and cytokine-induced neutrophil chemoattractant-1 levels, as well as total protein content, and decreased histologic indices of injury. These results demonstrate that aerosolized AAV vectors encoding human IκBα significantly attenuate endotoxin-mediated lung injury and may be a potential therapeutic candidate in the treatment of acute lung injury.

Optimized aerosol delivery to a mechanically ventilated rodent.

BACKGROUND: Aerosol delivery through an endotracheal tube during mechanical ventilation of small animals, simulating neonates and small infants, has shown to be influenced by a variety of factors including aerosol generator type, droplet/particle size, ventilator circuitry and ventilation regime. A review of the literature indicates that reported aerosol deposition rates in rodents are quite low, with lung deposition in anesthetized, mechanically ventilated rats reported to be approximately 3.9 and approximately 8% in anesthetized, spontaneously breathing rats. The optimization of aerosol delivery to both in vitro and in vivo models of anesthetized mechanically ventilated rodents is described in this study. METHODS: Characterization and optimization of the in vitro system performance relied on gravimetric analysis, laser diffraction droplet sizing, and spectrophotometric analysis of drug mass on inspiratory filters. The optimized setup was subsequently employed in vivo to determine deposition of a tracer aerosol in the rat lung. RESULTS: In vitro testing confirmed that droplet size, ventilation regimen, breath actuation setting, and the inclusion of a drug recycling step had the greatest effect on inhaled mass. During testing, improvements of up to 41% were seen in inhaled mass values between runs with the addition of a recycling step. The negative effects of the aerosolization process on albuterol sulphate were minimal. In vitro deposition rates of 29.95 +/- 1.54% of the original dose were recorded (n = 3). In vivo deposition rates of Evans blue were highly comparable (30.88 +/- 5.73%) (n = 6). Intratracheal instillation of the tracer dye resulted in deposition of 87.34 +/- 6.23% of the original dose. CONCLUSIONS: This optimized experimental setup allows for greater inhaled mass than previously reported. The addition of a recycling step may prove to be a significant improvement in achieving higher deposition in mechanically ventilated lungs; however, the suitability of the test agent for repeated nebulization needs assessment.