Inhalation Delivery Research Articles

Optimizing aerosolization efficiency of dry-powder aggregates of thermally-sensitive polymeric nanoparticles produced by spray-freeze-drying

Dry powder inhaler (DPI) delivery of therapeutic nanoparticles requires the nanoparticles to be transformed into inhalable micro-scale aggregate structures (i.e. nano-aggregates). The present work details the spray-freeze-drying (SFD) production of dry-powder aggregates of thermally-sensitive polymeric nanoparticles. Specifically, the aim is to optimize the aerosolization efficiency of the nano-aggregates, while keeping the morphology, production yield, flowability, and aqueous reconstitution in the desirable range. For this purpose, the effects of SFD process parameters (i.e. atomization rate, feed concentration, and feed rate) and freeze-drying adjuvant formulation on the nano-aggregate characteristics are examined. Low melting-point poly (caprolactone) (PCL) nanoparticles are used as the model nanoparticles. Mannitol and leucine are used as the hydrophilic and hydrophobic adjuvants, respectively. Large spherical porous nano-aggregates, where PCL nanoparticles are physically dispersed in the porous adjuvant matrix, have been produced. The presence of mannitol is crucial in ensuring that the nano-aggregates readily reconstitute into individual nanoparticles upon exposure to an aqueous environment, so that they can perform their intended therapeutic functions. The presence of leucine, on the other hand, is mandatory to obtain high aerosolization efficiency as its presence reduces the nano-aggregate tendency to agglomerate. At the optimal condition, nano-aggregates exhibiting ED (Emitted Dose) ≈ 95%, FPF (Fine Particle Fraction) ≈ 30%, and MMAD (Mass Median Aerodynamic Diameter) ≈ 5.3 μm, which are comparable to the values obtained in commercial DPI, have been produced. The results signify the potential of SFD to be employed in the production of inhalable dosage form of thermally-sensitive therapeutic nanoparticles.

Lung vascular targeting through inhalation delivery: Insight from filamentous viruses and other shapes

Systemic delivery of therapeutic agents via inhalation of particulates remains an attractive, noninvasive means of administration due to the possibilities of high bioavailability and high patient compliance. Optimization of particle shapes and particle properties for deep lung deposition after inhalation continues to be one of the key challenges. Here, we review several aspects of nanoparticle design for deep lung deposition as well as the nature and extent of translocation through the air-blood barrier for local or systemic vascular targeting. We describe filamentous influenza virus in comparison to worm-like "filomicelle" polymers as one example of a nature inspired design.

Pulmonary and Nasal Anti-Inflammatory and Anti-Allergy Inhalation Aerosol Delivery Systems

Most respiratory infections, diseases and allergic reactions have varying degrees of inflammation. Inflammation is a natural immunodefensive response to the presence of allergens or foreign particles that come into contact or affect the cells and tissues within the respiratory tract. The three main types of therapeutic drug classes available for anti-inflammatory and anti-allergy effects are corticosteroids, antihistamines and decongestants. Corticosteroid drugs for pulmonary inhalation and/or nasal delivery include beclomethasone dipropionate, budesonide, ciclesonide, fluticasone furoate, fluticasone propionate, mometasone furoate, and triamcinolone acetonide. Antihistamine drugs for nasal delivery include azelastine and olopatadine. Two common decongestants available are oxymetazoline and phenylephrine. Another therapeutic class, the anticholinergic agents, such as ipratropium bromide and tiotropium bromide, are used in pulmonary delivery in the treatment of inflammatory diseases such as asthma and chronic obstructive pulmonary disease. The mast cell stabilizer therapeutic class, cromolyn sodium, can be used to prevent and relieve nasal allergic symptoms. Additionally cromolyn sodium was the first dry powder inhaler product for pulmonary drug delivery several decades ago and currently is on the market as a pressurized metered dose inhaler for pulmonary inhalation delivery. Based on the devices used in pulmonary drug delivery, this route can be subdivided into three categories; namely nebulizers, pressurized metered dose inhalers, and dry powder inhalers. Nasal delivery of anti-inflammatory and antiallergy drugs is most commonly available commercially in an aqueous spray form. This article comprehensively reviews and discusses different kinds of drugs used for anti-inflammatory and anti-allergic effects via the pulmonary and nasal delivery route, as well as their mechanisms of action, marketed products, disease state indications while highlighting drug delivery and therapeutic aspects.

The twin impinger: a simple device for assessing the delivery of drugs from metered dose pressurized aerosol inhalers.

The development is described of the twin impinger, a two-stage separation device for assessing the drug delivery from metered dose inhalers and other oral inhalation delivery devices. The discharged aerosol is fractionated by firing through a simulated oropharynx and then through an impinger stage of defined aerodynamic particle size cut-off characteristics. The fine (pulmonary) fraction which penetrates is collected by a lower impinger. It is demonstrated that this device is able to assess individually the fine particle delivery of both components of two-drug aerosols. Formulations showing undue agglomeration or serious crystal growth of drug are readily detected. The twin impinger is shown to be a valuable device for routine quality assessment of aerosols during product development, stability testing and for quality assurance and comparison of commercial products.

Albumin-Coated Porous Hollow Poly(Lactic-co-Glycolic Acid) Microparticles Bound with Palmityl-Acylated Exendin-4 as a Long-Acting Inhalation Delivery System for the Treatment of Diabetes

To study the development of porous poly(lactic-co-glycolic acid) microparticles (PLGA MPs) coated initially with albumin and then with palmityl-acylated exendin-4 (Pal-Ex4) as an inhalation system for treating diabetes. Porous PLGA MPs were prepared by w/o/w double emulsification using hydroxypropyl-β-cyclodextrin and poly(ethylene-alt-maleic anhydride). Albumin was covalently attached to the MPs using EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide); Pal-Ex4 was then bound on the albumin surface. Albumin-binding degree and aerosolization efficiency were investigated. Deposition of the MPs after insufflations into the lungs of ICR mice was observed by image monitoring, and pulmonary hypoglycemic efficacies were examined in db/db mice. Cytotoxicity and histopathology induced by MPs were examined in Calu-3 and A549 cells and in the lungs of db/db mice, respectively. Albumin-coating and Pal-Ex4-binding to porous MP were performed with acceptable efficiencies. Pal-Ex4-bound albumin-coated MPs (Pal-Ex4/HSA-PLGA MP) were of high porosity and had appropriate aerodynamic sizes. Furthermore, this MP was efficiently deposited throughout mouse lungs, and exhibited a prolonged hypoglycemia and no significant lung tissue damage in db/db mice. Pal-Ex4/HSA-PLGA MP demonstrated many meaningful pharmaceutical advantages for the treatment of diabetes, in terms of aerosolization efficiency, drug loading, sustained drug-release, and hypoglycemic duration in vivo.

Formulations for Pulmonary Administration of Anticancer Agents to Treat Lung Malignancies

Chemotherapy plays a significant role both as primary and as supportive care for lung cancer treatment. The majority of currently available anticancer agents are administrated intravenously, causing side effects due to the systemic drug distribution. Alternatively, the bioavailability of orally administrated anticancer agents is usually compromised by the first-pass metabolism. Pulmonary administration may be a potential route for anticancer drug delivery to treat lung tumors, due to its site specific delivery, avoidance of first-pass metabolism, possibility of fewer side effects, and improved comfort for cancer patients using a needle-free delivery device. However, to attain an effective inhalational delivery, there is a requirement to design a formulation with appropriate aerodynamic properties with well-suited excipients. This review article explores work to date related to the formulations developed for pulmonary delivery of small molecule antineoplastic agents to treat primary and metastatic lung carcinomas. Ultimately, it highlights the importance of formulation design to define the role of inhalational chemotherapy.

Challenges and advances in the development of inhalable drug formulations for cystic fibrosis lung disease

Introduction: Cystic fibrosis (CF) is a multisystem genetic disorder, which usually results in significant respiratory dysfunction. At present there is no cure for CF, but advances in pharmacotherapy have gradually increased the life expectancy of CF patients. As many drugs used in the therapy of CF are delivered by inhalation, the demand for effective and convenient inhalational CF drug formulations will grow as CF patients live longer. Knowledge of the current limitations in inhalational CF drug delivery is critical in identifying new opportunities and designing rational delivery strategies.Areas covered: This review discusses current and emerging therapeutic agents for CF therapy, selected physiological challenges to effective inhalational medication delivery, and various approaches to overcoming these challenges. The reader will find an integrated view of the known inhalational drug delivery challenges and the rationales for recent investigational inhalational drug formulations.Expert opinion: An ideal drug/gene delivery system to CF airways should overcome the tenacious sputum, which presents physical, chemical and biological barriers to effective transport of therapeutic agents to the targets and various cellular challenges.

Mannitol‐Guided delivery of ciprofloxacin in artificial cystic fibrosis mucus model

Abnormal airway mucus presents a significant challenge for inhalational drug delivery. Recognizing the thick and tenacious airway mucus seen in the cystic fibrosis (CF) patients as a critical barrier to effective drug delivery, both into the mucus layer itself as well as across that layer to the underlying airway epithelium, we hypothesize that mannitol or NaCl can form inhalable drug carriers, improve drug penetration into the mucus, and ultimately enhance the drug's therapeutic effect. The objective of this study is to test whether mannitol and NaCl particles, as inhalable drug carriers, improve drug delivery into and enhance a drug's activity within a mucus-like material. Microparticles containing Ciprofloxacin (Cipro), an active ingredient, and either mannitol or NaCl were produced by spray-drying. Cipro encapsulated in mannitol particles (Cipro-mannitol) was significantly more effective at killing Pseudomonas aeruginosa (P. aeruginosa) grown in artificial mucus (AM) than Cipro encapsulated in either NaCl particles (Cipro-NaCl) or in hydrophobic particles consisting of dipalmitoylphosphatidylcholine (DPPC), albumin, and lactose (Cipro-DAL). The relatively high antibacterial effectiveness of Cipro-mannitol was not due to the effect of mannitol on bacteria or on Cipro. Rather, the unique performance of the mannitol-based particles in AM is attributable to its ability to increase local water content in the AM and enhance drug penetration into it. Mannitol is a promising excipient for inhalable microparticles that facilitate the drug delivery into the CF mucus.

Highly porous large poly(lactic-co-glycolic acid) microspheres adsorbed with palmityl-acylated exendin-4 as a long-acting inhalation system for treating diabetes

A porous large poly(lactic-co-glycolic acid) (PLGA) microspheres (MS) adsorbed with palmityl-acylated exendin-4 (Ex4-C 16) was devised as an inhalation delivery system. The porous MS was prepared by a single o/w emulsification/solvent evaporation method using extractable Pluronic F68/F127, and its fabrication and formulation conditions were carefully optimized. Results show that the prepared MS was in the appropriate size range for inhalation and contained many surfaces and internal pores meaning low aerodynamic density. Ex4-C 16 was more efficiently adsorbed onto porous PLGA MSs than native exendin-4, and an approximately 5% loading of Ex4-C 16 onto this porous MS (RG504H) was achieved. This optimized porous MS was found to be efficiently deposited throughout the entire lungs of mice including alveoli region. Furthermore, this porous MS adsorbed with Ex4-C 16 (approx. 100 μg/mouse) displayed much protracted hypoglycemic efficacy in non-fasted type 2 diabetic db/db mice. Porous PLGA MS with adsorbed Ex4-C 16 showed the dual-advantages of (i) sustained release and acceptable drug-loading due to strong hydrophobic interaction and (ii) longer in vivo pulmonary hypoglycemic duration due to albumin-binding by the palmityl group. We consider that this new prototype of porous PLGA MS has considerable pharmaceutical potential as a type 2 anti-diabetic inhalation treatment.

Battling With Environments: Drug Delivery to Target Tissues With Particles and Functional Biomaterials

Recent years have seen a growing interest in drug-delivery technology as an enabling tool for complicated pharmacological activities. At the same time, this field has faced as many challenges as successes in translating novel ideas into clinical benefits. The Laboratory for Therapeutic Particles and Biomaterials Engineering at Purdue University (IN, USA) has striven to identify the current challenges in drug delivery and find solutions through the design of new drug-delivery systems. We develop new inhalable formulations for drug and gene delivery for cystic fibrosis patients, simple particle platforms for inhalable drug delivery, anion-resistant nonviral gene vectors, tumor-targeted nanoparticle systems, and hydrogel-based therapeutics. Through expanded collaborations with researchers in medicine and related disciplines, we strive to contribute to advancing the drug delivery field in a clinically relevant manner.

Inhalation Delivery of a Novel Diindolylmethane Derivative for the Treatment of Lung Cancer

The purpose of this study was to determine the anticancer efficacy of 1,1-bis (3'-indolyl)-1-(p-biphenyl) methane (DIM-C-pPhC₆H₅) by inhalation delivery alone and in combination with i.v. docetaxel in a murine model for lung cancer. An aqueous DIM-C-pPhC₆H₅ formulation was characterized for its aerodynamic properties. Tumor-bearing athymic nude mice were exposed to nebulized DIM-C-pPhC₆H₅, docetaxel, or combination (DIM-C-pPhC₆H₅ plus docetaxel) using a nose-only exposure technique. The aerodynamic properties included mass median aerodynamic diameter of 1.8 ± 0.3 μm and geometric SD of 2.31 ± 0.02. Lung weight reduction in mice treated with the drug combination was 64% compared with 40% and 47% in mice treated with DIM-C-pPhC₆H₅ aerosol and docetaxel alone, respectively. Combination treatment decreased expression of Akt, cyclin D1, survivin, Mcl-1, NF-κB, IκBα, phospho-IκBα, and vascular endothelial growth factor (VEGF) and increased expression of c-Jun NH₂-terminal kinase 2 and Bad compared with tumors collected from single-agent treatment and control groups. DNA fragmentation was also enhanced in mice treated with the drug combination compared with docetaxel or DIM-C-pPhC₆H₅ alone. Combination treatment decreased expressions of VEGF and CD31 compared with single-agent treated and control groups. These results suggest that DIM-C-pPhC₆H₅ aerosol enhanced the anticancer activity of docetaxel in a lung cancer model by activating multiple signaling pathways. The study provides evidence that DIM-C-pPhC₆H₅ can be used alone or in combination with other drugs for the treatment of lung cancer using the inhalation delivery approach.

Dry powder measles vaccine: Particle deposition, virus replication, and immune response in cotton rats following inhalation

A stable and high potency dry powder measles vaccine with a particle size distribution suitable for inhalation was manufactured by CO2-Assisted Nebulization with a Bubble Dryer® (CAN-BD) process from bulk liquid Edmonston-Zagreb live attenuated measles virus vaccine supplied by the Serum Institute of India. A novel dry powder inhaler, the PuffHaler® was adapted for use in evaluating the utility of cotton rats to study the vaccine deposition, vaccine virus replication, and immune response following inhalation of the dry powder measles vaccine. Vaccine deposition in the lungs of cotton rats and subsequent viral replication was detected by measles-specific RT-PCR, and viral replication was confined to the lungs. Inhalation delivery resulted in an immune response comparable to that following injection. The cotton rat model is useful for evaluating new measles vaccine formulations and delivery devices.

A comparison between intratracheal and inhalation delivery of Aspergillus fumigatus conidia in the development of fungal allergic asthma in C57BL/6 mice

Allergic asthma is a debilitating disease of the airways characterized by airway hyperresponsiveness, eosinophilic inflammation, goblet cell metaplasia with associated mucus hypersecretion, and airway wall remodelling events, particularly subepithelial fibrosis and smooth muscle cell hyperplasia. Animal models that accurately mimic these hallmarks of allergic airways disease are critical for studying mechanisms associated with the cellular and structural changes that lead to disease pathogenesis. Aspergillus fumigatus, is a common aeroallergen of human asthmatics. The intratracheal (IT) delivery of A. fumigatus conidia into the airways of sensitized mice has been described as a model of allergic disease. Here, we compared the IT model with a newly developed inhalation (IH) challenge model. The IH model allowed multiple fungal exposures, which resulted in an exacerbation to the allergic asthma phenotype. Increased recruitment of eosinophils and lymphocytes, the hallmark leukocytes of asthma, was noted with the IH model as compared to the IT model in which macrophages and neutrophils were more prominent. Immunoglobulin E (IgE) production was significantly greater after IH challenge, while that of IgG2a was higher after IT challenge. Airway wall remodelling was pronounced in IH-treated mice, particularly after multiple allergen challenges. Although the IT model may be appropriate for the examination of the played by innate cells in the acute response to fungus, it fails to consistently reproduce the chronic remodelling hallmarks of allergic asthma. The ability of the IH challenge to mimic these characteristics recommends it as a model suited to study these important events.

Prediction of Efficacious Inhalation Lung Doses via the Use of In Silico Lung Retention Quantitative Structure-Activity Relationship Models and In Vitro Potency Screens

Lung concentrations of a drug are expected to drive the pharmacodynamic response to local inflammation after inhalation delivery, and the only way of determining the efficacious dose has been to measure it directly in animal models. In this study, we present a method to predict efficacious lung doses after inhalation in a rat lipopolysaccharide challenge model from in silico predictions of lung concentration and in vitro measurements only. A quantitative structure-activity relationship (QSAR) model, based on calculated physical properties predicted the partitioning of 34 compounds between lung and plasma. Because it was observed that lung/plasma partitioning correlated with lung concentration, it was possible to use this relationship to predict lung concentration at a given dose and time point. Based on the pharmacokinetic-pharmacodynamic (PKPD) relationship observed, a minimal free lung concentration relative to potency to drive significant inhibition of neutrophilia was established. By using predicted lung concentrations, measured fraction unbound in plasma, and cellular potency, it was possible to estimate an inhaled lung dose that would be expected to achieve this target exposure. These predictions were made for 23 compounds, which were not part of the original QSAR training set, and all except one were predicted to within 3-fold of their measured values. This novel approach shows that by understanding PKPD relationships and drivers for lung affinity after inhalation dosing, it is possible to estimate in vivo lung doses required for efficacy. This methodology provides a useful screening tool to rank candidate compounds and minimizes the use of extensive animal testing.

Pharmaceutical Potential of Gelatin as a pH-responsive Porogen for Manufacturing Porous Poly(d,l-lactic-co-glycolic acid) Microspheres

【Porous poly(lactic-co-glycolic acid) microspheres (PLGA MS) have been utilized as an inhalation delivery system and a matrix scaffold system for tissue engineering. Here, gelatin (type A) is introduced as an extractable pH-responsive porogen, which is capable of controlling the porosity and pore size of PLGA microspheres. Porous PLGA microspheres were prepared by a water-in-oil-in-water ( $w_1/o/w_2$ ) double emulsification/solvent evaporation method. The surface morphology of these microspheres was examined by varying pH (2.0~11.0) of water phases, using scanning electron microscopy (SEM). Also, their porosity and pore size were monitored by altering acidification time (1~5 h) using a phosphoric acid solution. Results showed that the pore-forming capability of gelatin was optimized at pH 5.0, and that the surface pore-formation was not significantly observed at pHs of 8.0. This was attributable to the balance between gel-formation by electrostatic repulsion and dissolution of gelatin. The appropriate time-selection between PLGA hardening and gelatin-washing out was considered as a second significant factor to control the porosity. Delaying the acidification time to ~5 h after emulsification was clearly effective to make pores in the microspheres. This finding suggests that the porosity and pore size of porous microspheres using gelatin can be significantly controlled depending on water phase pH and gelatin-removal time. The results obtained in this study would provide valuable pharmaceutical information to prepare porous PLGA MS, which is required to control the porosity.】

Inhalation Adjuvant Therapy for Lung Cancer

Lung cancer is the leading cause of new cancer cases and also is the number one cause of cancer death in both male and females in the United States and the world. Lung cancer is histologically separated into either small-cell (SCLC) or nonsmall-cell lung cancer (NSCLC). NSCLC is much more common, and can occur in the periphery (adenocarcinomas) or central airways (squamous cell carcinomas). The literature pertaining to lung cancer adjuvant therapy and inhalation lung cancer chemoprevention was reviewed. It is argued that inhalation of chemotherapy as an adjuvant in Stage II NSCC and inhalation of chemopreventive agents in Stage I adenocarcinomas are direct paths to improve lung cancer therapy as well as demonstrate the utility of inhalation therapy. If successful, inhalation delivery may be extended to the treatment of other types of lung cancer.

Evaluation of Inhaler Device Technique in Caregivers of Young Children with Asthma

Inhalation therapy has become the primary treatment mode for most pediatric pulmonary diseases. Errors in inhalation technique can decrease intrapulmonary drug deposition, which may lead to a decrease in therapeutic response. This study examined the ability of caregivers of young children with asthma, after being provided careful and standardized instruction, to correctly demonstrate how to use 1 of 2 different inhaler delivery systems. Caregivers of children aged 1–6 years, who were being prescribed an inhaler device for the first time, were trained by a staff asthma educator using a standardized educational checklist. One week later, the caregiver was required to demonstrate the correct preparation, use, and maintenance of their prescribed device to a different staff asthma educator, who rated their performance using the same standardized educational checklist. Errors (total and major) in carrying out the individual steps for each delivery device were recorded, and a Performance Mastery Score (measure of ability to do device steps correctly) was calculated. The total and major error rates were 24.8% and 15.6%, respectively, for the metered-dose inhalers (MDIs) with spacer, and 15.9% and 8.5%, respectively, for the nebulizer (P < 0.001). The mean Performance Mastery Score was 84.5% for the MDI with spacer and 91.9% for the nebulizer (P < 0.001). Caregivers of young children with asthma demonstrate a number of errors in device use, including major ones that can potentially result in poor lung delivery, even 1 week after standardized training by an asthma educator. Mastery of the MDI with spacer delivery system appears somewhat harder than that seen with the nebulizer. These findings suggest the need for repetition of sessions reinforcing inhalation instructions at follow-up in addition to initial education of proper device use, no matter what type of delivery system is selected.

Inhibition of lung tumor growth by complex pulmonary delivery of drugs with oligonucleotides as suppressors of cellular resistance

Development of cancer cell resistance, low accumulation of therapeutic drug in the lungs, and severe adverse treatment side effects represent main obstacles to efficient chemotherapy of lung cancer. To overcome these difficulties, we propose inhalation local delivery of anticancer drugs in combination with suppressors of pump and nonpump cellular resistance. To test this approach, nanoscale-based delivery systems containing doxorubicin as a cell death inducer, antisense oligonucleotides targeted to MRP1 mRNA as a suppressor of pump resistance and to BCL2 mRNA as a suppressor of nonpump resistance, were developed and examined on an orthotopic murine model of human lung carcinoma. The experimental results show high antitumor activity and low adverse side effects of proposed complex inhalatory treatment that cannot be achieved by individual components applied separately. The present work potentially contributes to the treatment of lung cancer by describing a unique combinatorial local inhalation delivery of drugs and suppressors of pump and nonpump cellular resistance.

An inhalation model of airway allergic response to inhalation of environmentalAspergillus fumigatusconidia in sensitized BALB/c mice

Fungal exposure may elicit a number of pulmonary diseases in man, including allergic asthma. Fungal sensitization is linked to asthma severity, although the basis for this increased pathology remains ambiguous. To create conditions simulating environmental fungal allergen exposure in a human, nose-only inhalation delivery of Aspergillus fumigatus conidia was employed in mice previously sensitized to Aspergillus antigen extract. BALB/c mice were immunized with subcutaneous and intraperitoneal injections of soluble A. fumigatus extract in alum, which was followed by three intranasal inoculations of the same fungal antigens dissolved in saline to elicit global sensitization in a manner similar to other published models. The animals were then challenged with a 10-min inhaled dose of live conidia blown directly from the surface of a mature A. fumigatus culture. After a single challenge with inhaled A. fumigatus conidia, allergic pulmonary inflammation and airway hyperresponsiveness were significantly increased above that of either naïve animals or animals that had been sensitized to A. fumigatus antigens but not challenged with conidia. The architecture of the lung was changed by inhalation of conidia when compared to controls in that there were significant increases in epithelial thickness, goblet cell metaplasia, and peribronchial collagen deposition. Additionally, α-smooth muscle actin staining of histological sections showed visual evidence of increased peribronchial smooth muscle mass after fungal challenge. In summary, the delivery of live A. fumigatus conidia to the sensitized airways of BALB/c mice advances the study of the pulmonary response to fungi by providing a more natural route of exposure and, for the first time, demonstrates the consistent development of fibrosis and smooth muscle changes accompanying exposure to inhaled fungal conidia in a mouse model.

RNAi-mediated suppression of constitutive pulmonary gene expression by small interfering RNA in mice

The ability of synthetic small interfering RNA (siRNA) to silence gene expression makes it a useful tool in biomedical research. However, effective and non-toxic functional siRNA delivery to mouse lungs in vivo is still a key challenge, and regulation of constitutively expressed genes is poorly characterized. Following in vitro validation of siRNA molecules, naked, stabilized siRNA (AtuRNAi) was applied intranasally (i.n.) by droplets or intratracheally (i.t.) by MicroSprayer™ in female C57BL/6 mice. Distribution of Cy3-tagged siRNAs was examined. Pulmonary expression of ubiquitously (lamin B1) or cell-specific (E-cadherin, VE-cadherin), constitutive genes was analysed by TaqMan-realtime-PCR. Further, formulated lipoplex-siRNA, which has enhanced transfection efficiency, was applied i.t. or intravenously (i.v.). Single i.t. as compared to i.n. application of unformulated siRNA resulted in higher delivery efficiency and homogenous pulmonary distribution. After inhalation of target-specific siRNA, reduction of epithelial E-cadherin by 21%, but no significant reduction of endothelial VE-cadherin or ubiquitously expressed lamin B1 was observed. Pharmacokinetic analysis revealed rapid transfer of intact siRNA molecules into the vascular system and accumulation in the kidneys, calling lung specificity into question. I.t. application of lipoplex-siRNA evoked inflammation. In contrast, i.v. application of lipoplex-siRNA specifically reduced expression of VE-cadherin mRNA by about 50% in lungs without evoking lung cellular influx. In conclusion, sufficient pulmonary distribution of aerosolized siRNA was attained in mice by MicroSprayer™, however development of appropriate siRNA carriers is highly desirable to improve lung-specific functional inhalative siRNA delivery. Pulmonary knockdown of constitutive endothelial targets by 50% was achieved by i.v. application of lipoplex-siRNA.