Exploring the Toxicity, Lung Distribution, and Cellular Uptake of Rifampicin and Ascorbic Acid-Loaded Alginate Nanoparticles as Therapeutic Treatment of Lung Intracellular Infections.

Nanotechnology is a very promising technological tool to combat health problems associated with the loss of effectiveness of currently used antibiotics. Previously, we developed a formulation consisting of a chitosan and tween 80-decorated alginate nanocarrier that encapsulates rifampicin and the antioxidant ascorbic acid (RIF/ASC), intended for the treatment of respiratory intracellular infections. Here, we investigated the effects of RIF/ASC-loaded NPs on the respiratory mucus and the pulmonary surfactant. In addition, we evaluated their cytotoxicity for lung cells in vitro, and their biodistribution on rat lungs in vivo after their intratracheal administration. Findings herein demonstrated that RIF/ASC-loaded NPs display a favorable lung biocompatibility profile and a uniform distribution throughout lung lobules. RIF/ASC-loaded NPs were mainly uptaken by lung macrophages, their primary target. In summary, findings show that our novel designed RIF/ASC NPs could be a suitable system for antibiotic lung administration with promising perspectives for the treatment of pulmonary intracellular infections.

C-Kit+ progenitors restore rat asthmatic lung function by modulation of T-bet and GATA-3 expression.

NEW FINDINGS: What is the central question of this study? The aim of the experiment was to highlight the regenerative capacity of bone marrow Kit+ cells in the restoration of asthmatic pulmonary function in the rat model. What is the main finding and its importance? Data showed that these cells were recruited successfully to the asthmatic niche after intratracheal administration and accelerated the regeneration of asthmatic lungs by the modulation of inflammation via the control of Gata3 and Tbx21 expression, leading to decreased tracheal responsiveness to methacholine and reduction of pathological remodelling. ABSTRACT: Allergic asthma is a T helper (Th) 2 immunological disorder with consequential uncontrolled inflammatory responses. There is an increasing demand to use new methods for the treatment of asthma based on modulation of the Th2-to-Th1 ratio in favour of the Th1 population. Accordingly, we decided to evaluate the effects of intratracheal administration of Kit+ bone marrow cells on tracheal responsiveness and the expression of Gata3 and Tbx21 genes. Forty male Wistar rats were allocated  randomly  into four experimental groups: healthy rats (control group), sensitized rats (OVA group), sensitized rats receiving Kit- cells (OVA+Kit- group) and sensitized rats receiving Kit+ cells (OVA+Kit+ group). Total and differential white blood cell counts, tracheal responsiveness to cumulative methacholine concentrations and histopathological analysis were evaluated. The results showed a statistically significant increase in total white blood cell, eosinophil and neutrophil counts, tracheal contractility, Gata3 expression and prototypical histopathology of asthma. Along with these conditions, we found that the number of lymphocytes was decreased and expression of Tbx21 diminished in sensitized rats compared with control animals. Monitoring of labelled tagged cells confirmed successful engraftment of transplanted cells in pulmonary tissue. Juxtaposition of Kit+ cells changed the blood leucogram closer to the control values. Kit+ cells increased the expression of Tbx21 and suppressed Gata3 (P < 0.05). In the OVA+Kit+ group, tracheal responsiveness was improved coincident with increased pulmonary regeneration. In conclusion, this study showed that intratracheal administration of bone marrow-derived Kit+ cells, but not Kit- cells, could be effective in the alleviation of asthma, presumably by the modulation of Gata3 and Tbx21.

A review of pulmonary toxicity studies of nanocellulose.

In recent years, nanocellulose (NC) obtained by defibrating cellulose to the nanometer level has been developed, and its development for various applications, e.g. as an additive for cosmetics and as a component of structural elements, is progressing. However, because NC has unique physico-chemical properties that are not found in conventional nanomaterials, particularly when inhaled, there are concerns about unexpected effects on organisms. This review summarizes the progress of in vivo experiments on the effects of NC on the respiratory system by inhalation. In addition, this review will provide new insights into NC toxicity studies by comparing the effects of fibrous nanomaterials.

Local pulmonary drug delivery in the preterm rabbit: feasibility and efficacy of daily intratracheal injections.

Recent clinical trials in newborns have successfully used surfactant as a drug carrier for an active compound, to minimize systemic exposure. To investigate the translational potential of surfactant-compound mixtures and other local therapeutics, a relevant animal model is required in which intratracheal administration for maximal local deposition is technically possible and well tolerated. Preterm rabbit pups (born at 28 days of gestation) were exposed to either hyperoxia or normoxia and randomized to receive daily intratracheal surfactant, daily intratracheal saline, or no injections for 7 days. At day 7, the overall lung function and morphology were assessed. Efficacy in terms of distribution was assessed by micro-PET-CT on both day 0 and day 7. Lung function as well as parenchymal and vascular structure were altered by hyperoxia, thereby reproducing a phenotype reminiscent of bronchopulmonary dysplasia (BPD). Neither intratracheal surfactant nor saline affected the survival or the hyperoxia-induced BPD phenotype of the pups. Using PET-CT, we demonstrate that 82.5% of the injected radioactive tracer goes and remains in the lungs, with a decrease of only 4% after 150 min. Surfactant and saline can safely and effectively be administered in spontaneously breathing preterm rabbits. The described model and method enable researchers to evaluate intratracheal pharmacological interventions for the treatment of BPD.

Pharmacokinetics and Pharmacodynamics of Nebulized and Intratracheal Milrinone in a Swine Model of Hypercapnia Pulmonary Hypertension.

OBJECTIVES: Milrinone pulmonary administration is used currently for the treatment of pulmonary hypertension. Several methods are available: simple jet nebulization, vibrating mesh nebulization, intratracheal instillation, and intratracheal atomization. The aim of this study was to explore the concentration-effect relationship of milrinone for each of these methods. DESIGN: Observational open-label pharmacokinetic/pharmacodynamics cohort study. SETTING: Single-center, hospital animal laboratory. PARTICIPANTS: Twelve swine. INTERVENTIONS: After hypercapnia pulmonary hypertension, swine were administered equivalent inhaled milrinone doses of 15 or 50 µg/kg through simple jet nebulization, vibrating mesh nebulization, intratracheal instillation, and intratracheal atomization. MEASUREMENTS AND MAIN RESULTS: Blood and urine samples were taken up to 360 minutes postadministration. The ratio of mean systemic arterial pressure to mean pulmonary arterial pressure was monitored continuously. Right heart echographies were taken before and after hypercapnia and after drug administration. Mean elimination half-lives were similar for the 4 administrations. Mean percent changes in the ratio were of 37 (60%), 18 (31%), 17 (36%), and 20 (20%), for simple jet nebulization, vibrating mesh nebulization, intratracheal instillation, and intratracheal atomization, respectively. Mean maximum plasma concentrations for intratracheal administrations were reached at 8 and 45 min for atomization and instillation, respectively. Significant increases in right atrial diameter and right ventricular end-diastolic area were witnessed during pulmonary hypertension as well as a return to baseline values after milrinone administration. CONCLUSIONS: The intratracheal methods, particularly intratracheal atomization, showed less hemodynamic effect than nebulizations and, in the case of intratracheal instillation, unpredictable drug exposure. Nebulization methods on the other hand, especially simple jet nebulization, suggest better efficacy and sensitivity but are less fit for emergency situations.

Pulmonary delivery of polyplexes for combined PAI-1 gene silencing and CXCR4 inhibition to treat lung fibrosis.

This report describes the development of polyplexes based on CXCR4-inhibiting poly(ethylenimine) derivative (PEI-C) for pulmonary delivery of siRNA to silence plasminogen activator inhibitor-1 (siPAI-1) as a new combination treatment of pulmonary fibrosis (PF). Safety and delivery efficacy of the PEI-C/siPAI-1 polyplexes was investigated in vitro in primary lung fibroblasts isolated from mice with bleomycin-induced PF. Biodistribution analysis following intratracheal administration of fluorescently labeled polyplexes showed prolonged retention in the lungs. Treatment of mice with bleomycin-induced PF using the PEI-C/siPAI-1 polyplexes resulted in a significant down-regulation of the PAI-1 expression and decreased collagen deposition in the lung. The results of this study provide first evidence of the potential benefits of combined inhibition of CXCR4 and PAI-1 in the pulmonary treatment of PF.

Experimental Substantiation of Inhalation Administration of Pathogenic Therapy of Toxic Convulsive Disorder for Correction of External Respiration Disorders.

We studied the dynamics of respiratory function in rats during intratracheal poisoning with diisopropyl fluorophosphate and pentylenetetrazole in doses corresponding to the LD50 in humans. The maximum of external respiration impairment was recorded in 30 min after poisoning. Administration of diazepam and atropine both separately and in combination during the development of the first signs of poisoning did not significantly affect the respiration parameters, but reduced the incidence of seizures and contributed to a decrease in the rate of animal death. Intratracheal administration of cholinolytic, ß2-adrenomimetic, or glutamate receptors antagonist promoted correction of the respiratory function. It was found that the maximum therapeutic effect in case of diisopropyl fluorophosphates poisoning was achieved after intratracheal administration of ipratropium bromide (0.086 mg/kg), salbutamol (0.086 mg/kg), and MK-801 (0.1 mg/kg), while in case of pentylenetetrazole poisoning, intratracheal administration of ipratropium bromide (0.086 mg/kg) was most effective.

Pharmacokinetics/Pharmacodynamics of Pulmonary Delivery of Colistin against Pseudomonas aeruginosa in a Mouse Lung Infection Model.

Colistin is often administered by inhalation and/or the parenteral route for the treatment of respiratory infections caused by multidrug-resistant (MDR) Pseudomonas aeruginosa However, limited pharmacokinetic (PK) and pharmacodynamic (PD) data are available to guide the optimization of dosage regimens of inhaled colistin. In the present study, PK of colistin in epithelial lining fluid (ELF) and plasma was determined following intratracheal delivery of a single dose of colistin solution in neutropenic lung-infected mice. The antimicrobial efficacy of intratracheal delivery of colistin against three P. aeruginosa strains (ATCC 27853, PAO1, and FADDI-PA022; MIC of 1 mg/liter for all strains) was examined in a neutropenic mouse lung infection model. Dose fractionation studies were conducted over 2.64 to 23.8 mg/kg of body weight/day. The inhibitory sigmoid model was employed to determine the PK/PD index that best described the antimicrobial efficacy of pulmonary delivery of colistin. In both ELF and plasma, the ratio of the area under the unbound concentration-time profile to MIC (fAUC/MIC) was the PK/PD index that best described the antimicrobial effect in mouse lung infection (R2 = 0.60 to 0.84 for ELF and 0.64 to 0.83 for plasma). The fAUC/MIC targets required to achieve stasis against the three strains were 684 to 1,050 in ELF and 2.15 to 3.29 in plasma. The histopathological data showed that pulmonary delivery of colistin reduced infection-caused pulmonary inflammation and preserved the integrity of the lung epithelium, although colistin introduced mild pulmonary inflammation in healthy mice. This study showed pulmonary delivery of colistin provides antimicrobial effects against MDR P. aeruginosa lung infections superior to those of parenteral administrations. For the first time, our results provide important preclinical PK/PD information for optimization of inhaled colistin therapy.

Kinetics and dissolution of intratracheally administered nickel oxide nanomaterials in rats.

BACKGROUND: The toxicokinetics of nanomaterials are an important factor in toxicity, which may be affected by slow clearance and/or distribution in the body. METHODS: Four types of nickel oxide (NiO) nanoparticles were single-administered intratracheally to male F344 rats at three doses of 0.67-6.0 mg/kg body weight. The rats were sacrificed under anesthesia and the lung, thoracic lymph nodes, bronchoalveolar lavage fluid, liver, and other organs were sampled for Ni burden measurement 3, 28, and 91 days post-administration; Ni excretion was measured 6 and 24 h after administration. Solubility of NiO nanoparticles was determined using artificial lysosomal fluid, artificial interstitial fluid, hydrogen peroxide solution, pure water, and saline. In addition, macrophage migration to trachea and phagosome-lysosome-fusion rate constants were estimated using pulmonary clearance and dissolution rate constants. RESULTS: The wire-like NiO nanoparticles were 100% dissolved by 24 h when mixed with artificial lysosomal fluid (dissolution rate coefficient: 0.18/h); spherical NiO nanoparticles were 12% and 35% dissolved after 216 h when mixed with artificial lysosomal fluid (1.4 × 10-3 and 4.9 × 10-3/h). The largest irregular-shaped NiO nanoparticles hardly dissolved in any solution, including artificial lysosomal fluid (7.8 × 10-5/h). Pulmonary clearance rate constants, estimated using a one-compartment model, were much higher for the NiO nanoparticles with a wire-shape (0.069-0.078/day) than for the spherical and irregular-shaped NiO nanoparticles (0-0.012/day). Pulmonary clearance rate constants of the largest irregular-shaped NiO nanoparticles showed an inverse correlation with dose. Translocation of NiO from the lungs to the thoracic lymph nodes increased in a time- and dose-dependent manner for three spherical and irregular-shaped NiO nanoparticles, but not for the wire-like NiO nanoparticles. Thirty-five percent of the wire-like NiO nanoparticles were excreted in the first 24 h after administration; excretion was 0.33-3.6% in that time frame for the spherical and irregular-shaped NiO nanoparticles. CONCLUSION: These findings suggest that nanomaterial solubility differences can result in variations in their pulmonary clearance. Nanoparticles with moderate lysosomal solubility may induce persistent pulmonary inflammation.

Comparison of the local pulmonary distribution of nanoparticles administered intratracheally to rats via gavage needle or microsprayer delivery devices.

Intratracheal administration methods are used to conduct toxicological assessments of inhaled nanoparticles (NPs), and gavage needles or microsprayers are common intratracheal delivery devices. The NP suspension is delivered in a liquid state via gavage needle and as a liquid aerosol via microsprayer. The differences in local pulmonary NP distribution (called the microdistribution) arising from the different states of the NP suspension cause differential pulmonary responses; however, this has yet to be investigated. Herein, using microbeam X-ray fluorescence microscopy, we quantitatively evaluated the TiO2 pulmonary microdistribution (per mesh: 100 µm × 100 µm) in lung sections from rats administered an intratracheal dose of TiO2 NPs (6 mg kg-1 ) via gavage needle or microsprayer. The results revealed that: (i) using a microsprayer appears to reduce the variations in TiO2 content (ng mesh-1 ) among rats (e.g., coefficients of variation, n = 3, microsprayer vs gavage needle: 13% vs 30%, for the entire lungs); (ii) TiO2 appears to be deposited less in the right middle lobes than in the rest of the lung lobes, irrespective of the chosen intratracheal delivery device; and (iii) similar TiO2 contents (ng mesh-1 ) and frequencies are deposited in the lung lobes of rats administered TiO2 NPs via gavage needle or microsprayer. This suggests that the physical state of the administered NP suspension does not markedly alter TiO2 pulmonary microdistribution. The results of this investigation are important for the standardization of intratracheal administration methods. Copyright © 2016 John Wiley & Sons, Ltd.

Intratracheal Milrinone Bolus Administration During Acute Right Ventricular Dysfunction After Cardiopulmonary Bypass.

OBJECTIVE: To evaluate intratracheal milrinone (tMil) administration for rapid treatment of right ventricular (RV) dysfunction as a novel route after cardiopulmonary bypass. DESIGN: Retrospective analysis. SETTING: Single-center study. PARTICIPANTS: The study comprised 7 patients undergoing cardiac surgery who exhibited acute RV dysfunction after cardiopulmonary bypass. INTERVENTIONS: After difficult weaning caused by cardiopulmonary bypass-induced acute RV dysfunction, milrinone was administered as a 5-mg bolus inside the endotracheal tube. MEASUREMENTS AND MAIN RESULTS: RV function improvement, as indicated by decreasing pulmonary artery pressure and changes of RV waveforms, was observed in all 7 patients. Adverse effects of tMil included dynamic RV outflow tract obstruction (2 patients) and a decrease in systemic mean arterial pressure (1 patient). CONCLUSIONS: tMil may be an effective, rapid, and easily applicable therapeutic alternative to inhaled milrinone for the treatment of acute RV failure during cardiac surgery. However, sufficiently powered clinical trials are needed to confirm these findings.

Fibrotic gene expression coexists with alveolar proteinosis in early indium lung.

Occupational inhalation of indium compounds can cause the so-called "indium lung disease". Most affected individuals show pulmonary alveolar proteinosis (PAP) and fibrotic interstitial lung disease. In animal experiments, inhalation of indium tin oxide or indium oxide has been shown to cause lung damage. However, the mechanisms by which indium compounds lead to indium lung disease remain unknown. In this study, we constructed a mouse model of indium lung disease and analyzed gene expression in response to indium exposure. Indium oxide (In2O3, 10 mg/kg, primary particle size <100 nm) was administered intratracheally to C57BL/6 mice (male, 8 weeks of age) twice a week for 8 weeks. Four weeks after the final instillation, histopathological analysis exhibited periodic acid-Schiff positive material in the alveoli, characteristic of PAP. Comprehensive gene expression analysis by RNA-Seq, however, revealed expression of fibrosis-related genes, such as surfactant associated protein D, surfactant associated protein A1, mucin 1, and collagen type I and III, was significantly increased, indicating that fibrotic gene expression progresses in early phase of indium lung. These data supported the latest hypothesis that PAP occurs as an acute phase response and is replaced by fibrosis after long-term latency.

Effects of dose volume and delivery device on bronchoalveolar lavage parameters of intratracheally administered nano-sized TiO2 in rats.

The intratracheal (IT) test is useful for screening the pulmonary toxicity of inhaled materials, including nanomaterials. However, a standard procedure has not yet been authorized internationally, and the effects of different test parameters are unknown. To determine appropriate experimental conditions for the IT test, we intratracheally administered nano-sized TiO2 to male F344 rats at 3.0 mg/kg body weight by using two delivery devices (gavage needle or microaerosolizer) and dose volumes of 0.5-3.0 mL/kg (gavage needle) or 0.5-2.0 mL/kg (microaerosolizer). We evaluated the pulmonary deposition and interlobar distribution of TiO2 at both 30 min and 3 days after administration. In addition, the inflammatory components in bronchoalveolar lavage (BAL) fluid were measured 3 days after administration of TiO2. At dose volumes of 0.5-2.0 mL/kg, the BAL values were comparable regardless of the device used. In addition, pulmonary TiO2 burden and lobar concentration patterns were equivalent at all combinations of dose volume and delivery device. In conclusion, the acute pulmonary toxicity of nanomaterials can be assessed effectively by using an IT test in which the test agent is provided to rats at a dose volume of 0.5-2.0 mL/kg with either a gavage needle or microaerosolizer.

Quantitative evaluation of local pulmonary distribution of TiO2 in rats following single or multiple intratracheal administrations of TiO2 nanoparticles using X-ray fluorescence microscopy.

Uneven pulmonary nanoparticle (NP) distribution has been described when using single-dose intratracheal administration tests. Multiple-dose intratracheal administrations with small quantities of NPs are expected to improve the unevenness of each dose. The differences in local pulmonary NP distribution (called microdistribution) between single- and multiple-dose administrations may cause differential pulmonary responses; however, this has not been evaluated. Here, we quantitatively evaluated the pulmonary microdistribution (per mesh: 100 µm × 100 µm) of TiO2 in lung sections from rats following one, two, three, or four doses of TiO2 NPs at a same total dosage of 10 mg kg(-1) using X-ray fluorescence microscopy. The results indicate that: (i) multiple-dose administrations show lower variations in TiO2 content (ng mesh(-1) ) for sections of each lobe; (ii) TiO2 appears to be deposited more in the right caudal and accessory lobes located downstream of the administration direction of NP suspensions, and less so in the right middle lobes, irrespective of the number of doses; (iii) there are not prominent differences in the pattern of pulmonary TiO2 microdistribution between rats following single and multiple doses of TiO2 NPs. Additionally, the estimation of pulmonary TiO2 deposition for multiple-dose administrations imply that every dose of TiO2 would be randomly deposited only in part of the fixed 30-50% of lung areas. The evidence suggests that multiple-dose administrations do not offer remarkable advantages over single-dose administration on the pulmonary NP microdistribution, although multiple-dose administrations may reduce variations in the TiO2 content for each lung lobe. Copyright © 2016 John Wiley & Sons, Ltd.

Histological Quantification of Gene Silencing by Intratracheal Administration of Dry Powdered Small-Interfering RNA/Chitosan Complexes in the Murine Lung.

PURPOSE: The use of small-interfering RNA (siRNA) as an inhalation therapy has recently received much attention. Some reports have confirmed the suppression of gene expression in whole lungs following intratracheal administration of dry powdered siRNA; however, the anatomical location in the lung where gene silencing occurs has not been precisely identified. Here, we aimed to histologically evaluate gene silencing efficacy in murine lungs by intratracheal administration of an siRNA/chitosan complex as a dry powder. METHODS: Enhanced green fluorescence protein (EGFP)-specific siRNA (EGFP-siRNA)/chitosan powder was prepared and administered intratracheally to EGFP transgenic mice or mice carrying metastatic lung tumors consisting of Lewis lung carcinoma (LLC) cells stably expressing EGFP (EGFP-LLCs). Thereafter, green fluorescence intensities were quantified in the airways, parenchyma, and lung tumors. RESULTS: Intratracheal administration of the EGFP-siRNA/chitosan powder suppressed EGFP expression in the bronchi, bronchioles, and alveolar walls of EGFP transgenic mice. Additionally, EGFP-siRNA/chitosan effectively silenced EGFP expression in lung tumors consisting of EGFP-LLC cells. CONCLUSIONS: Pulmonary administration of siRNA/chitosan powder suppressed gene expression throughout the lung and in lung tumors. Therefore, this may become a powerful strategy to target genes expressed in a wide range of respiratory diseases involving the airways, parenchyma, and lung tumors.

Quantitative evaluation of the pulmonary microdistribution of TiO2 nanoparticles using X-ray fluorescence microscopy after intratracheal administration with a microsprayer in rats.

The unevenness of pulmonary nanoparticle (NP) distribution, which hinders the establishment of an absolute dose-response relationship, has been described as one of the limitations of intratracheal administration techniques for toxicological assessment of inhaled NPs. Quantification of the NP microdistribution would facilitate the establishment of a concentration-response relationship in localized regions of the lung; however, such quantitative methods have not been reported. Here, we established a quantitative method for evaluating pulmonary TiO2 NP microdistribution in rats using X-ray fluorescence microscopy. Ti intensity in lung sections from rats intratracheally administered 10 mg kg(-1) TiO2 NPs with a microsprayer was measured using X-ray fluorescence with a 100 µm beam size. Ti reference samples were prepared by dropping different concentrations of Ti solutions on glass slide or lung sections of untreated rat. Ti intensity increased linearly with Ti content in the reference samples on both substrates. The detection limit of TiO2 was estimated to be 6.3 ng mm(-2) . The reproducibility was confirmed for measurements done in the short- (2 weeks) and long-term (6 months). The quantitative results of TiO2 NP microdistribution suggested that more TiO2 NPs were distributed in the right caudal and accessory lobes, which are located downstream of the administration direction of the NP suspension, and the lower portion of each lobe. The detection rates of TiO2 NPs were 16.6-25.0%, 5.19-15.6%, 28.6-39.2%, 21.4-38.7% and 10.6-23.2% for lung sections from the right cranial, middle, caudal, accessory and left lobes, respectively.

Intratracheal administration of programmable DNA nanostructures combats acute lung injury by targeting microRNA-155.

Acute lung injury (ALI) is an acute inflammatory process that can result in life-threatening consequences. Programmable DNA nanostructures have emerged as excellent nanoplatforms for microRNA-based therapeutics, offering potential nanomedicines for ALI treatment. Nonetheless, the traditional systematic administration of nanomedicines is constrained by low delivery efficiency, poor pharmacokinetics, and nonspecific side effects. Here, we identify macrophage microRNA-155 as a novel therapeutic target using the magnetic bead sorting technique. We further construct a DNA nanotubular nucleic acid drug antagonizing microRNA-155 (NT-155) for ALI treatment through intratracheal administration. Flow cytometry results demonstrate that NT-155, when inhaled, is taken up much more effectively by macrophages and dendritic cells in the bronchoalveolar lavage fluid of ALI mice. Furthermore, NT-155 effectively silences the overexpressed microRNA-155 in macrophages and exerts excellent inflammation inhibition effects in vitro and ALI mouse models. Mechanistically, NT-155 suppresses microRNA-155 expression and activates its target gene SOCS1, inhibiting the p-P65 signaling pathway and suppressing proinflammatory cytokine secretion. The current study suggests that deliberately designed nucleic acid drugs are promising nanomedicines for ALI treatment and the local administration may open up new practical applications of DNA in the future.

Inhibition of influenza virus infection in mice by pulmonary administration of a spray dried antiviral.

Increasing resistance to antiviral drugs approved for the treatment of influenza urges the development of novel compounds. Ideally, this should be complemented by a careful consideration of the administration route. 6'siallyllactosamine-functionalized ß-cyclodextrin (CD-6'SLN) is a novel entry inhibitor that acts as a mimic of the primary attachment receptor of influenza, sialic acid. In this study, we aimed to develop a dry powder formulation of CD-6'SLN to assess its in vivo antiviral activity after administration via the pulmonary route. By means of spray drying the compound together with trileucine, a dispersion enhancer, we created a powder that retained the antiviral effect of the drug, remained stable under elevated temperature conditions and performed well in a dry powder inhaler. To test the efficacy of the dry powder drug against influenza infection in vivo, infected mice were treated with CD-6'SLN using an aerosol generator that allowed for the controlled administration of powder formulations to the lungs of mice. CD-6'SLN was effective in mitigating the course of the disease compared to the control groups, reflected by lower disease activity scores and by the prevention of virus-induced IL-6 production. Our data show that CD-6'SLN can be formulated as a stable dry powder that is suitable for use in a dry powder inhaler and is effective when administered via the pulmonary route to influenza-infected mice.

Influence of lipid composition of messenger RNA-loaded lipid nanoparticles on the protein expression via intratracheal administration in mice.

Intratracheal (i.t.) administration, which takes advantage of the specific structure of the respiratory system, can effectively deliver nanoparticles to the lung. Much remains unknown about the i.t. administration of messenger RNA (mRNA)-lipid nanoparticles (LNPs) and the effect of lipid composition. In this study, we administered minute amounts of mRNA-LNP solutions into mice intratracheally and investigated the effect of lipid composition on protein expression in the lungs. We first validated higher protein expression with mRNA-LNP compared to that with mRNA-PEI complex and naked mRNA. Then, we evaluated the influence of lipid composition of LNPs on the protein expression and found that: 1) decreasing the PEG molarity from 1.5% to 0.5% could significantly increase the protein expression; 2) replacing DMG-PEG with DSG-PEG could slightly increase the protein expression; 3) using DOPE instead of DSPC could increase protein expression by an order of magnitude. We successfully prepared an mRNA-LNP with optimal lipid compositions that led to robust protein expression following i.t. administration, thus providing meaningful insights into advanced development of mRNA-LNPs for therapeutic i.t. administration.

Performance Testing of a Homemade Aerosol Generator for Pulmonary Administration of Dry Powder Formulations to Mice.

A challenge in the development of dry powder formulations for inhalation is the poor reproducibility of their administration to small laboratory animals. The currently used devices for the pulmonary administration of dry powder formulations to small rodents often function sub-optimally as they use the same puff of air for both powder dispersion and aerosol delivery. As a result, either the air volume and flow rate are too low for complete powder deagglomeration or they are too high for effective aerosol delivery to the lungs of the animal. Therefore, novel and better devices are desired. We here present an aerosol generator designed to administer a pre-generated aerosol to the lungs of mice. By mapping the complex relationship between the airflow rate, delivery time and emitted dose, we were able to control the amount of powder being delivered from the aerosol generator. The emitted aerosol had a size range favorable for lung deposition and could be measured reproducibly. Nevertheless, in vivo fluorescent imaging still revealed considerable differences between the mice in terms of the dose deposited and the distribution of powder over the lungs, suggesting that a certain biological variation in lung deposition is inevitable.